CN108503724B - Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof - Google Patents
Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof Download PDFInfo
- Publication number
- CN108503724B CN108503724B CN201810389126.0A CN201810389126A CN108503724B CN 108503724 B CN108503724 B CN 108503724B CN 201810389126 A CN201810389126 A CN 201810389126A CN 108503724 B CN108503724 B CN 108503724B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- cordyceps militaris
- culture medium
- solution
- militaris culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- 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
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Toxicology (AREA)
- Sustainable Development (AREA)
- Epidemiology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physical Education & Sports Medicine (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a cordyceps militaris culture medium polysaccharide, a separation and purification method and application thereof, wherein the polysaccharide consists of monosaccharides in the following molar percentage: 0.26% ribose, 0.11% rhamnose, 2.01% arabinose, 0.34% xylose, 29.62% mannose, 67.19% glucose, 0.47% galactose. The extraction method does not affect the biological activity of the cordyceps militaris culture medium polysaccharide P2, and the obtained pure polysaccharide P2 has high purity and stable property, has obvious effects on oxidation resistance, uric acid reduction and bacteriostasis, and is beneficial to human metabolism; the cost is low, and the pure product of the polysaccharide P2 can be further used for the development of health care products, medicines and cosmetics.
Description
Technical Field
The invention relates to cordyceps militaris culture medium polysaccharide and a separation and purification method and application thereof.
Background
Polysaccharides (polysaccharides) are also called polysaccharides, linear or branched chain polymers formed by connecting aldoses or ketoses together through glycosidic bonds are polar complex macromolecules with the polymerization degree of more than 10, the molecular weight is generally more than tens of thousands, the polysaccharides are one of four basic substances forming vital activities, and the polysaccharides in organisms can be combined with proteins or fats to form proteoglycan and lipopolysaccharide besides existing in a free state.
Cordyceps militaris (C.militaris) is also called as Cordyceps militaris, belongs to Ascomycotina, Pyrenomycetes, and Mycosphaerellales in taxonomic manner, belongs to the same genus as Cordyceps militaris in the genus of Maillariaceae, and is mainly distributed in northeast, northwest, and northwest regions of China. The Cordyceps militaris can parasitize larvae or pupae of lepidoptera, coleopteran, diptera and other insects, and can be artificially cultured in batch by using silkworm pupae, rice culture medium and the like.
Because natural cordyceps militaris resources are limited, at present, artificial cultivation is more, and three culture methods are mainly adopted: (1) collecting wild Cordyceps militaris, separating and purifying strains, inoculating the Cordyceps militaris strains on a solid culture medium such as rice containing pupa Bombycis powder, and culturing at certain temperature, humidity and illumination for 35-45d to obtain Cordyceps militaris fruiting body; (2) inoculating Cordyceps militaris strain in silkworm larva or living pupa, and culturing at certain temperature, humidity and illumination for 35-45d to obtain Cordyceps militaris fruiting body; (3) the soybean meal sucrose or corn steep liquor sucrose is used as a culture medium, and the cordyceps militaris is cultured by liquid fermentation.
The production of artificially cultured cordyceps militaris sporocarp in China has been carried out on a considerable scale, but a large amount of cordyceps militaris culture medium leftovers are produced while the sporocarp is harvested, so that not only is the environment polluted, but also the resource waste is not negligible.
Research shows that the cordyceps militaris culture medium leftovers contain bioactive substances such as cordycepin, cordyceps polysaccharide and the like. Therefore, the utilization, research and development of the cordyceps militaris culture medium leftovers are necessary for better developing and utilizing cordyceps militaris resources, improving economic benefits and perfecting the cordyceps militaris industrial chain.
Chinese patent application No. 201110086808.2 discloses a method for extracting polysaccharide from Cordyceps militaris culture medium, which comprises removing protein and starch from Cordyceps militaris culture medium with different enzyme solutions, and precipitating with ethanol to obtain Cordyceps militaris culture medium crude polysaccharide; the operation process is complex, the cost is high, the polysaccharide cannot be further separated and purified, and the polysaccharide component with high purity cannot be obtained.
Disclosure of Invention
The invention aims to provide cordyceps militaris culture medium polysaccharide.
The invention also aims to provide the separation and purification method of the cordyceps militaris culture medium polysaccharide, which is characterized in that the cordyceps militaris waste culture medium is subjected to polysaccharide extraction by using an ultrasonic-assisted water extraction and alcohol precipitation method, and the polysaccharide is separated and purified by using an ion exchange chromatography method, so that polysaccharide components with higher purity and bioactivity are extracted and separated.
The invention also aims to provide application of the cordyceps militaris culture medium polysaccharide.
The purpose of the invention is realized by the following technical scheme:
a cordyceps militaris culture medium polysaccharide is composed of the following monosaccharides in mole percentage: 0.26% ribose, 0.11% rhamnose, 2.01% arabinose, 0.34% xylose, 29.62% mannose, 67.19% glucose, 0.47% galactose;
the average molecular weight of the cordyceps militaris culture medium polysaccharide is 16.6k Da;
the cordyceps militaris culture polysaccharide contains pyranose rings and beta-type glycosidic bonds.
The separation and purification method of the cordyceps militaris culture medium polysaccharide comprises the following steps:
(1) extracting cordyceps militaris culture medium polysaccharide: drying and crushing the cordyceps militaris rice culture medium leftovers, and sieving to obtain leftover dry powder; weighing dry powder, adding 15-16 times of distilled water, performing ultrasonic treatment for more than 30min, reflux-extracting at 70 deg.C for 1.5-2.0h, extracting for several times, mixing extractive solutions, filtering, and concentrating to obtain polysaccharide concentrated solution; adding 95% (V/V) ethanol with volume 3-4 times of that of the polysaccharide concentrated solution, stirring, and standing at 4 ℃ overnight; centrifuging and drying the precipitate to obtain a cordyceps militaris culture substrate polysaccharide extract;
sieving in the step (1) is preferably carried out by a 40-mesh sieve;
the concentration in step (1) is preferably at 50-55 ℃;
the centrifugation of the step (1) is preferably carried out for 15min at 5000 r/min;
(2) decoloring cordyceps militaris culture medium polysaccharide: dissolving Cordyceps militaris culture medium polysaccharide extract with distilled water, adjusting pH to 8.0-8.5, and adding dropwise H2O2The solution is colorless, and the temperature is kept at 50-55 ℃ for more than 2 h;
h in the step (2)2O2A solution, preferably at a concentration of 30% (V/V);
(3) the enzyme method is combined with Sevage method to remove protein:
3-1: mixing the papain solution and the cordyceps militaris culture-based polysaccharide extracting solution according to the volume ratio of 1.0: 1.5-1.0: 1.7, and carrying out enzymolysis for 2-3h at the temperature of 60-70 ℃;
the papain solution is prepared by PBS buffer solution with the pH value of 6.0, wherein the concentration of the papain is preferably 250U/ml;
3-2: adding a Sevage reagent with the volume of 1/5 into the enzymolysis liquid, carrying out shaking culture for more than 30min, then centrifuging for multiple times until no protein precipitate is separated out, taking supernatant, and drying to obtain cordyceps militaris culture medium crude polysaccharide;
the Sevage reagent in the step (3) is prepared from chloroform and n-butanol according to the volume ratio of 5: 1;
performing shaking culture in the step (3), wherein the rotating speed of a shaking table is preferably 150 r/min;
the centrifugation of the step (3) is preferably carried out for 20-30min at 4000 r/min;
(4) separating and purifying cordyceps militaris culture medium polysaccharide: dissolving the crude polysaccharide of the cordyceps militaris culture medium by using distilled water, then putting the solution on a DEAE sepharose FF (DEAE Sepharose Fast flow) ion exchange chromatographic column, and eluting the solution by using 0.1mol/L NaCl solution to obtain the polysaccharide P2 of the cordyceps militaris culture medium.
The separation and purification of the polysaccharide not only is a process for removing impurities, but also is a process for separating the mixed polysaccharide into single components. The column chromatography can be classified into ion exchange chromatography and gel column chromatography. Ion exchange chromatography is a fractionation based on the difference in ion charge density, with anion exchangers having positively charged groups and negatively charged counterions, so that the exchangers can exchange reactions with negatively charged compounds or anions in solution, and cation exchangers vice versa.
The cordyceps militaris culture medium polysaccharide can be applied as an antioxidant or a bacteriostatic agent, and can also be used for preparing a medicament with the effect of reducing uric acid.
The invention obtains the cordyceps militaris culture medium crude polysaccharide by a water extraction and alcohol precipitation method, and the separation and purification of the crude polysaccharide are carried out by using an ion exchange chromatographic column, compared with the prior art, the method has the following advantages and effects:
(1) the invention fully utilizes the culture medium leftovers generated by culturing the cordyceps militaris, accords with the green environmental protection concept of changing waste into valuable, establishes a complete and feasible technical route for researching the polysaccharide extraction, separation and purification, physicochemical properties, structure and biological activity of the cordyceps militaris culture medium, perfects the residue treatment process of the artificial culture cordyceps militaris industry, and provides technical guidance for the extraction, separation and purification of the edible fungus polysaccharide.
(2) The water extraction and alcohol precipitation method can complete the extraction operation of the polysaccharide with large flux, has lower cost, good repeatability and high yield, and is suitable for industrial large-scale production.
(3) The DEAE Sepharose Fast Flow used in the invention has better physical and chemical stability and mechanical property, large exchange capacity, can be cleaned in place, has small change of bed volume along with the ionic strength of pH, and is suitable for purifying a large amount of crude products due to high Flow rate and loading capacity.
(4) The extraction method does not affect the biological activity of the cordyceps militaris culture medium polysaccharide P2, and the obtained pure polysaccharide P2 has high purity and stable property, has obvious effects on oxidation resistance, uric acid reduction and bacteriostasis, and is beneficial to human metabolism; the cost is low, and the pure product of the polysaccharide P2 can be further used for the development of health care products, medicines and cosmetics.
(5) The invention creatively combines the water extraction and alcohol precipitation extraction method of the polysaccharide with the ion exchange chromatography separation and purification method for researching the cordyceps militaris culture medium leftover polysaccharide, compares and obtains better process parameters, and determines DEAE Sepharose Fast Flow as a chromatographic column filler, thereby providing technical guidance and a new idea for the extraction, separation and purification of the cordyceps militaris culture medium leftover polysaccharide.
Drawings
FIG. 1 is the elution profile of polysaccharide P2 from Cordyceps militaris culture medium.
FIG. 2 is a UV spectrum of polysaccharide P2.
FIG. 3 is an infrared spectrum of polysaccharide P2.
FIG. 4 shows the ABTS free radical scavenging ability of polysaccharide P2.
FIG. 5 is a graph showing the results of the bacteriostatic test on polysaccharide P2; wherein, Sa-the inhibition zone for staphylococcus aureus, Pa-the inhibition zone for pseudomonas aeruginosa, 2-the inhibition zone generated by polysaccharide P2, and 0-the inhibition zone of the control group.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
In the invention, the analysis of the physicochemical properties of the cordyceps militaris culture medium polysaccharide is carried out by the Guangzhou analysis and test center in China, and the report numbers are 2018001306-2b respectively.
Example 1
A method for extracting, separating and purifying polysaccharide from cordyceps militaris culture medium leftovers comprises the following steps:
(1) extracting cordyceps militaris culture medium polysaccharide: weighing 55g of cordyceps militaris rice culture medium leftovers, fully drying, crushing, sieving with a 40-mesh sieve, weighing 50g of dry powder, adding 800ml of distilled water, carrying out ultrasonic treatment for 30min, carrying out reflux extraction at 70 ℃ for 1.5h, extracting for 3 times, combining filtrates, carrying out vacuum filtration, and concentrating at 55 ℃ to 100ml to obtain polysaccharide concentrated solution; adding 400ml of 95% ethanol into the polysaccharide concentrated solution, continuously stirring to precipitate polysaccharide uniformly, and standing overnight at 4 deg.C; centrifuging at 5000r/min for 15min, and oven drying the precipitate to obtain Cordyceps militaris culture polysaccharide extract;
(2) decoloring cordyceps militaris culture medium polysaccharide: dissolving Cordyceps militaris culture medium polysaccharide extract with distilled water to obtain polysaccharide extractive solution with concentration of 0.05g/ml, adding NaOH to adjust pH to 8.0, and adding 30% H dropwise2O2Until colorless, and keeping the temperature at 50 ℃ for 2 h;
(3) the enzyme method is combined with Sevage method to remove protein:
accurately weighing 0.1g of papain, dissolving into a solution with a final concentration of 250U/ml by using PBS buffer solution with a pH value of 6.0, mixing with the cordyceps militaris culture medium polysaccharide extracting solution, carrying out enzymolysis for 3 hours at 64 ℃ with the volume ratio of the enzyme solution to the cordyceps militaris culture medium polysaccharide extracting solution being 1.0: 1.5;
adding 1/5 volume of Sevage reagent (chloroform: n-butanol is 5:1) into the enzymolysis solution, placing in a shaking table 150r/min, oscillating for 30min, centrifuging at 4000r/min for 20min, repeating the centrifugation for multiple times until no protein precipitate is separated out, combining the supernatants, and drying at 50 ℃ to obtain Cordyceps militaris culture medium crude polysaccharide;
(4) separating and purifying cordyceps militaris culture medium polysaccharide:
weighing 0.1g of cordyceps militaris culture medium crude polysaccharide, dissolving in 10ml of distilled water, loading on a DEAE Sepharose Fast Flow ion exchange chromatography column, eluting with 0.1mol/L NaCl solution at the Flow rate of 0.5ml/min, collecting 1 tube every 10min, detecting the polysaccharide content tube by a phenol-sulfuric acid method, and combining according to an elution curve (figure 1) to obtain the cordyceps militaris culture medium polysaccharide P2.
The concrete operation of detecting the polysaccharide content by a phenol-sulfuric acid method is as follows: accurately weighing 0.1g of anhydrous glucose standard substance dried to constant weight at 105 ℃, placing the anhydrous glucose standard substance in a 100ml volumetric flask, adding distilled water for dissolving, fixing the volume, shaking up, and preparing into a standard substance solution of 1mg/ml for later use. The solution is diluted into standard solutions with different concentrations of 10, 20, 40, 60, 80 and 100 mu g/ml respectively. Respectively sucking 1ml of the above solutions, placing in a test tube, adding 0.5ml of 6% phenol solution, mixing, adding 2.5ml of concentrated sulfuric acid, mixing, standing at room temperature for 20min, measuring absorbance at 490nm with distilled water as blank control, and drawing a standard curve with glucose concentration as abscissa and OD value as ordinate. The unknown sample is used for determining the polysaccharide content by a standard curve method.
Concentrating the eluted polysaccharide component, dialyzing, and freeze-drying to obtain pure Cordyceps militaris culture polysaccharide product named as P2.
Example 2
Performing ultraviolet spectrum analysis on the cordyceps militaris culture medium polysaccharide P2 obtained in example 1, weighing 1mg of polysaccharide sample respectively, preparing 1mg/mL of polysaccharide solution, and scanning an ultraviolet spectrum within the range of 200-800 nm.
FIG. 2 is a UV spectrum of P2, and the result shows that P2 has no obvious absorption peak at 260nm and 280nm, which indicates that P2 contains no protein and nucleic acid substances.
Example 3
Polysaccharide molecular weight analysis is carried out on the cordyceps militaris culture medium polysaccharide P2 obtained in example 1, and the specific experimental method is as follows:
the molecular weight was determined by Gel Permeation Chromatography (GPC). Weighing freeze-dried polysaccharide sample 2mg, adding 0.02M phosphate buffer solution for dissolving, preparing into 2.0mg/mL solution, filtering with 0.22 μ M sterile filter membrane, and collecting the filtrate for use.
Chromatographic conditions are as follows: the column temperature is 35 ℃; 0.02mol/L phosphate buffer solution (pH value 7.0) is used as a mobile phase, the flow rate is 0.6ml/min, and the sample volume is 20 mu L; TSK gel protection column (40mm × 6.0mm), TSKG-4000K gel column (300mm × 7.8mm) and TSKG-2500K gel column (300mm × 7.8 mm); waters 2414 shows differential refractive detector detection. A series of dextran solutions (700,400,200,100,50,30,10,5kD) of different molecular weights were prepared as standards and standard curves were made. The molecular weight of the samples was calculated against their corresponding elution volume against a standard curve.
The result shows that the average molecular weight of the cordyceps militaris culture medium polysaccharide P2 is 16.6k Da.
Example 4
Monosaccharide composition analysis is performed on the cordyceps militaris culture medium polysaccharide P2 obtained in example 1, and the specific method is as follows:
a10 mg sample of polysaccharide was weighed, added with 5mL of trifluoroacetic acid (4M) and hydrolyzed at 110 ℃ for 2 h. The hydrolysate was evaporated to dryness under vacuum at 50 ℃ and washed 3 times with chromatographically pure methanol (adding chromatographically pure methanol and spin-drying again, repeating 3 times until the dried material had no trifluoroacetic acid taste), to obtain polysaccharide hydrolysate.
Adding 10mg of hydroxylamine hydrochloride, 1mg of internal standard inositol and 2mL of pyridine into the polysaccharide hydrolysate in sequence, sealing, adding 2m L acetic anhydride after 30min of water bath at 90 ℃, and adding 2m L water to terminate the reaction. Extracting with 2m L dichloromethane, repeating for 2 times, mixing dichloromethane phases, adding anhydrous sodium sulfate, drying, and filtering with 0.22 μm organic microporous membrane.
The analysis was carried out by gas chromatography using an HP-5MS quartz capillary column (30 m. times.0.25 mm. times.0.25 μm). The temperature-raising program is as follows: the injection port temperature is 250 ℃, the initial column temperature is 100 ℃, and the temperature is kept for 0.5 min; then raising the temperature to 140 ℃ at the speed of 20 ℃/min, and keeping the temperature for 5 min; raising the temperature to 160 ℃ at the speed of 3 ℃/min; then the temperature is raised to 250 ℃ at the speed of 10 ℃/min and kept for 5 min. The sample injection volume is 1 mu L; the split ratio is 10: 1; the mobile phase is helium; the flow rate was 1 mL/min.
Various monosaccharide standards (rhamnose, arabinose, ribose, xylose, mannose, glucose and galactose) are tested according to the same steps, and the treated monosaccharide standard is analyzed by gas chromatography according to the same detection degree.
The results of measuring the monosaccharide composition of the cordyceps militaris culture medium polysaccharide are shown in the following table:
TABLE 1 monosaccharide composition of polysaccharide P2 in Cordyceps militaris culture medium
Example 5
Performing Fourier infrared spectrum analysis on the cordyceps militaris culture medium polysaccharide P2 obtained in example 1:
weighing 2mg polysaccharide sample, mixing with dried KBr (potassium bromide) in mortar, grinding, tabletting with tablet machine, and performing Fourier transform infrared spectrometer at 400-4000cm-1Is scanned over a range of wavenumbers.
FIG. 3 is an infrared spectrum of P2 at 3401cm-1The peak at (A) is generated by O-H stretching vibration of P2, and 2929cm-1The peak at (A) is generated by C-H vibration, 1642cm-1The peak at (b) is caused by C ═ O stretching vibration of P2, and these peaks are characteristic peaks of polysaccharides, indicating that P2 belongs to polysaccharides.
1153cm in the infrared spectrum of P2-1The peak is absorption peak of C-O on the ring, 1080, 1027cm-1The peak of (A) is generated by the variable angle vibration of the alcoholic hydroxyl group, and the three peaks indicate that pyranose ring exists in P2, 862cm-1The peak at (a) indicates the presence of a β -type glycosidic linkage in P2.
Example 6
The polysaccharide P2 in the cordyceps militaris culture medium obtained in example 1 is subjected to antioxidant capacity measurement:
ABTS free radical scavenging capacity assay:
mixing 5mL of 7mmol/L ABTS aqueous solution and 5mL of 2.45mmol/L potassium persulfate aqueous solution, placing the mixture in the dark for reaction for 12h to generate ABTS free radicals, and diluting the ABTS free radical solution to ensure that the absorbance value of the ABTS free radical solution under the condition of 734nm wavelength is 0.70 +/-0.02. Mixing 1mL of Cordyceps militaris culture medium polysaccharide P1 solution with different mass concentrations and 2mL of ABTS free radical solution, measuring absorbance at 734nm after 10min, and recording as A1(ii) a 2mL of the ABTS free radical solution was mixed with 1mL of distilled water, and the absorbance at 734nm was measured and recorded as A0(ii) a The absorbance at 734nm of 2mL of distilled water and 1mL of polysaccharide solution was measured and recorded as A2。
Clearance (%): y ═ 1- (A)1-A2)/A0]×100％
FIG. 4 shows ABTS free radical scavenging ability of polysaccharide P2 in Cordyceps militaris culture medium. As shown in FIG. 4, in the concentration range of 1.0-5.0 mg/ml, P2 has a certain ability of scavenging ABTS free radicals and is positively correlated with the polysaccharide concentration. The ABTS free radical clearance of P2 was 38.0% at a polysaccharide concentration of 5.0 mg/ml.
Example 7
Uric acid reduction study was performed on the cordyceps militaris culture medium polysaccharide P2 obtained in example 1:
(1) establishment of hyperuricemia model
60 male mice were bred for one week and then randomly divided into 6 groups of 10 mice each, which were a blank group, a model group, a polysaccharide P2 high (400mg/kg), a polysaccharide P2 medium (200mg/kg), a P2 low (100mg/kg) dose group, and a positive drug (50mg/kg) control group. 600mg/kg of xanthine (ig) + 100mg/kg of potassium oxonate (ip) is intraperitoneally administered by intragastric gavage in a morning model group, a positive control group and a polysaccharide P2 high, medium and low dose group every day. One hour before the model building, the water is not forbidden, and the blank group is given the same dosage of physiological saline. The high, medium and low dose groups of polysaccharide P2 at afternoon adopt polysaccharide suspension for intragastric administration, the positive control group adopts allopurinol suspension 50mg/kg for intragastric administration, the model group and the blank group adopt normal saline with the same volume for intragastric administration, and the determination of in vivo biochemical index is continuously carried out for seven days (2)
Weighing the body weight after 1h of administration in the afternoon on the seventh day, cutting off the head and taking blood, standing at room temperature for 40min, centrifuging at 3000r/min for 10min, sucking upper serum, detecting Uric Acid (UA), serum Creatinine (CREA) and serum urea nitrogen (BUN) values in the serum by using a kit, and inspecting the influence of polysaccharide on UA, CREA, BUN and renal function of a hyperuricemia model mouse.
The results of the uric acid lowering effect of the polysaccharide P2 in the cordyceps militaris culture medium are shown in the following table:
TABLE 2 uric acid lowering action of polysaccharide P2 in Cordyceps militaris culture medium
As can be seen from Table 2, compared with the normal group, the serum creatinine, serum uric acid and serum urea nitrogen levels of the model group mice are all significantly increased, indicating that the molding is successful.
Compared with the model group, the low, medium and high dose group of the polysaccharide P2 can respectively reduce the serum creatinine level of mice by 2.22%, 10.06% and 19.01%, can respectively reduce the serum uric acid level of mice by 3.52%, 11.16% and 19.80%, and can respectively reduce the serum urea nitrogen level of mice by 10.22%, 22.70% and 43.30%. The experimental model shows that the cordyceps militaris culture medium polysaccharide P2 can reduce serum creatinine, serum uric acid and serum urea nitrogen levels of hyperuricemia.
Example 8
The antibacterial study of the cordyceps militaris culture medium polysaccharide P2 obtained in example 1 was carried out:
the antibacterial activity of common pathogenic bacteria staphylococcus aureus (Sa) and pseudomonas aeruginosa (Pa) is determined by adopting a perforation method. The main operation process is as follows: taking a culture dish with the diameter of 90mm, pouring the plate, taking 150 mul of bacterial suspension by using a pipette, uniformly coating each plate, punching 4 holes by using an 8mm puncher, respectively adding 40 mul of 1mg/ml cordyceps militaris culture medium polysaccharide P2 for later use, and adding 40 mul of physiological saline into each hole of a control group. Culturing in a constant temperature incubator at 37 deg.C for 24h, measuring the diameter of each zone of inhibition on the experimental plate and the control plate respectively by cross method, and calculating the average value.
The results of the bacteriostatic experiment of the polysaccharide P2 are shown in FIG. 5, which are the bacteriostatic result of P2 on Staphylococcus aureus, the bacteriostatic result of P2 on Pseudomonas aeruginosa and the bacteriostatic result of Pseudomonas aeruginosa, respectively, and the diameters of the measured bacteriostatic circles are shown in Table 3.
TABLE 3 antibacterial Ring diameter (cm)
As can be seen from Table 3, the cordyceps militaris culture medium polysaccharide P2 has different degrees of inhibition effects on Staphylococcus aureus and Pseudomonas aeruginosa, wherein the inhibition effect on Staphylococcus aureus is stronger than that on Pseudomonas aeruginosa.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. The application of the cordyceps militaris culture substrate polysaccharide in the preparation of the staphylococcus aureus bacteriostat is characterized in that:
the cordyceps militaris culture medium polysaccharide is composed of the following monosaccharides in percentage by mole: 0.26% ribose, 0.11% rhamnose, 2.01% arabinose, 0.34% xylose, 29.62% mannose, 67.19% glucose, 0.47% galactose; the average molecular weight is 16.6k Da, and the polysaccharide compound contains pyranose rings and beta-type glycosidic bonds;
the separation and purification method of the cordyceps militaris culture medium polysaccharide comprises the following steps:
(1) extracting cordyceps militaris culture medium polysaccharide: drying and crushing the cordyceps militaris rice culture medium leftovers, and sieving to obtain leftover dry powder; weighing dry powder, adding 15-16 times of distilled water, performing ultrasonic treatment for more than 30min, reflux-extracting at 70 deg.C for 1.5-2.0h, extracting for several times, mixing extractive solutions, filtering, and concentrating to obtain polysaccharide concentrated solution; adding 95% V/V ethanol with volume 3-4 times of that of the polysaccharide concentrated solution, stirring, and standing at 4 deg.C overnight; centrifuging and drying the precipitate to obtain a cordyceps militaris culture substrate polysaccharide extract;
(2) decoloring cordyceps militaris culture medium polysaccharide: dissolving Cordyceps militaris culture medium polysaccharide extract with distilled water, adjusting pH to 8.0-8.5, and adding dropwise H2O2The solution is colorless, and the temperature is kept at 50-55 ℃ for more than 2 h;
(3) the enzyme method is combined with Sevage method to remove protein:
3-1: mixing the papain solution and the cordyceps militaris culture-based polysaccharide extracting solution according to the volume ratio of 1.0: 1.5-1.0: 1.7, and carrying out enzymolysis for 2-3h at the temperature of 60-70 ℃;
3-2: adding a Sevage reagent with the volume of 1/5 into the enzymolysis liquid, carrying out shaking culture for more than 30min, then centrifuging for multiple times until no protein precipitate is separated out, taking supernatant, and drying to obtain cordyceps militaris culture medium crude polysaccharide;
(4) separating and purifying cordyceps militaris culture medium polysaccharide: dissolving the crude polysaccharide of the cordyceps militaris culture medium by using distilled water, putting the solution on a DEAE sepharose FF ion exchange chromatography column, and eluting the solution by using 0.1mol/L NaCl solution to obtain the polysaccharide.
2. Use according to claim 1, characterized in that: the concentration in the step (1) is concentration at 50-55 ℃.
3. Use according to claim 1, characterized in that: h in the step (2)2O2Solution, concentration 30% V/V.
4. Use according to claim 1, characterized in that: and (3) preparing the papain solution in the step (3) by using PBS (phosphate buffer solution) with the pH value of 6.0, wherein the concentration of the papain is 250U/ml.
5. Use according to claim 1, characterized in that: the Sevage reagent in the step (3) is prepared from chloroform and n-butanol according to the volume ratio of 5: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810389126.0A CN108503724B (en) | 2018-04-27 | 2018-04-27 | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810389126.0A CN108503724B (en) | 2018-04-27 | 2018-04-27 | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108503724A CN108503724A (en) | 2018-09-07 |
| CN108503724B true CN108503724B (en) | 2021-06-01 |
Family
ID=63399499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810389126.0A Active CN108503724B (en) | 2018-04-27 | 2018-04-27 | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108503724B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109846909B (en) * | 2019-01-31 | 2022-03-22 | 武汉康复得生物科技股份有限公司 | Composition capable of catalyzing uric acid degradation in intestinal tract and preparation method and application thereof |
| CN114836501B (en) * | 2022-04-13 | 2023-08-22 | 深圳大学 | Silkworm chrysalis protein peptide with uric acid reducing and anti-inflammatory activities and preparation method and application thereof |
| CN116425901B (en) * | 2023-06-13 | 2023-08-18 | 西南民族大学 | Bitter bamboo shoot polysaccharide and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558264A (en) * | 2012-01-18 | 2012-07-11 | 辽宁仙榆湾北冬虫夏草（集团）有限公司 | Method for extracting cordycepin and cordyceps polysaccharide from cordyceps militaris |
| CN105085704A (en) * | 2015-09-15 | 2015-11-25 | 上海瑞丰农业科技有限公司 | Preparation method of cordyceps militaris active polysaccharide |
| CN105585639A (en) * | 2015-12-08 | 2016-05-18 | 泰山医学院 | Cordyceps militaris sporocarp heteropolysaccharide and application thereof |
| CN109748979A (en) * | 2017-11-02 | 2019-05-14 | 张松林 | A kind of extracting method of cordyceps flower polysaccharide |
-
2018
- 2018-04-27 CN CN201810389126.0A patent/CN108503724B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558264A (en) * | 2012-01-18 | 2012-07-11 | 辽宁仙榆湾北冬虫夏草（集团）有限公司 | Method for extracting cordycepin and cordyceps polysaccharide from cordyceps militaris |
| CN105085704A (en) * | 2015-09-15 | 2015-11-25 | 上海瑞丰农业科技有限公司 | Preparation method of cordyceps militaris active polysaccharide |
| CN105585639A (en) * | 2015-12-08 | 2016-05-18 | 泰山医学院 | Cordyceps militaris sporocarp heteropolysaccharide and application thereof |
| CN109748979A (en) * | 2017-11-02 | 2019-05-14 | 张松林 | A kind of extracting method of cordyceps flower polysaccharide |
Non-Patent Citations (4)
| Title |
|---|
| "Hypouricemic Actions of Exopolysaccharide Produced by Cordyceps militaris in Potassium Oxonate-Induced Hyperuricemic Mice";Li Ma等;《CURRENT MICROBIOLOGY》;20140803;第69卷(第6期);第852-857页 * |
| "蛹虫草乙醇分级多糖生理功能研究";陈宏伟等;《食品与发酵工业》;20080930;第34卷(第9期);第16-19页 * |
| "蛹虫草固体栽培及下脚料深加工工艺研究";王军;《中国优秀硕士学位论文全文数据库 农业科技辑》;20090815(第 08 期);第D047-134页 * |
| 王军."蛹虫草固体栽培及下脚料深加工工艺研究".《中国优秀硕士学位论文全文数据库 农业科技辑》.2009,(第 08 期),第D047-134页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108503724A (en) | 2018-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108546306B (en) | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof | |
| CN108503724B (en) | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof | |
| US11473115B2 (en) | Exopolysaccharide from Rhodopseudomonas palustris and method for preparing and use thereof | |
| CN103725730B (en) | A kind of process for refining of Hericium erinaceus (Bull. Ex Fr.) Pers. Rich in selenium mycelium polysaccharides | |
| CN104945527A (en) | Galactomannan antigen and preparation method thereof | |
| CN101492706A (en) | Method for improving cordyceps sinensis bacterium native volume of production with cordyceps militaris link liquid fermentation | |
| CN113274490A (en) | Preparation method and application of botryococcus longipediculus polysaccharide disease-resistant inducer | |
| CN1683418A (en) | Process for preparing antrodia camphorata polysaccharide and antrodia camphorata triterpene with micro-prorous adsorptive resin and its product | |
| CN113831419B (en) | Armillariella tabescens mycelium polysaccharide and its preparation method and bacteriostatic use | |
| CN108641006B (en) | Method for extracting, separating and purifying polysaccharide from cordyceps militaris culture medium leftovers | |
| CN112552424A (en) | Cordyceps sobolifera sporophore polysaccharide and preparation and application thereof | |
| Huang et al. | Preparation and antioxidant activity in vitro of fermented Tremella fuciformis extracellular polysaccharides | |
| CN1266164C (en) | Lucid ganoderma spore powder polysaccharide, production method and use | |
| JP2004091780A (en) | Polysaccharides of antrodiacamphorata fungus | |
| CN115869343A (en) | Application of Shandong Ganoderma extracellular ethanol precipitate in preparing antitumor drugs | |
| CN1123577C (en) | Ocean thalassiomycete hypoxylon polyose and its extracting process and application | |
| CN113651898A (en) | Coprinus comatus mycelium polysaccharide and preparation method and application thereof | |
| Kovalenko et al. | Glycans of higher Basidiomycetes mushrooms with antiphytoviral properties: isolation, characterization, and biological activity | |
| CN113244258A (en) | Preparation method and application of spirulina polysaccharide proinflammatory enzyme inducer | |
| CN114752641B (en) | Preparation method of Antrodia camphorata selenium polysaccharide | |
| CN100417390C (en) | Effective part of sweet wormwood, its preparation method and application thereof in pharmacy | |
| CN115947876B (en) | beta-D-galactoglucan and preparation and application thereof | |
| CN114409824B (en) | Mucor exopolysaccharide and preparation method and application thereof | |
| CN111704679B (en) | Beta-glucan of hericium erinaceus fermentation mycelium and preparation method and application thereof | |
| CN115109168B (en) | Gentiana rigescens neutral polysaccharide and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |