CN111172216A - Cordyceps militaris polysaccharide with function of inhibiting macrophage from secreting NO, and preparation method and application thereof - Google Patents
Cordyceps militaris polysaccharide with function of inhibiting macrophage from secreting NO, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cordyceps militaris polysaccharide with a function of inhibiting macrophage from secreting NO, and a preparation method and application thereof. The method comprises the following steps: inoculating Cordyceps militaris strains in a liquid culture medium for fermentation culture, and performing solid-liquid separation to obtain Cordyceps militaris fermentation liquor and Cordyceps militaris mycelia; then degreasing the mycelium, extracting with hot water, and concentrating to obtain a cordyceps militaris extraction concentrated solution; and then removing protein, precipitating with ethanol, decolorizing with trypsin and Sevage method, and purifying with DEAE-cellulose column chromatography and cross-linked Sephadex G-100 gel column to obtain intracellular polysaccharide and extracellular polysaccharide of Cordyceps militaris. The cordyceps militaris polysaccharide prepared by the method can effectively inhibit macrophage from secreting NO, has good anti-inflammatory effect, and can be used in the field of anti-inflammatory drugs or health-care food.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to cordyceps militaris polysaccharide with a function of inhibiting macrophage from secreting NO, and a preparation method and application thereof.
Background
The fermentation liquid and mycelium are obtained by submerged fermentation of edible and medicinal fungi liquid, and intracellular polysaccharide and extracellular polysaccharide can be respectively extracted from the mycelium. Cordyceps militaris (Cordyceps militaris), also known as Cordyceps militaris (L.) Link, is a typical large edible and medicinal fungus, and is parasitic on larva and mushroom fruiting body, and contains substances (Tuli, Hardeep S, Sharma, Anil K, Sandhu, Sardul S, et al. Cordycepin: A bioactive metabolism with therapeutic potential [ J ]. Life Sciences,2013,93(23):863-869.) with high nutritive value and medicinal value. As a traditional Chinese medicine, Cordyceps militaris has pharmacological effects such as antioxidation (Cordyceps fungi: natural products, pharmacological functions and developmental products [ J ]. Journal of pharmacy & Pharmacology,2010,61(3):279-291.), immunomodulation (Lull C, Wichers H J, Savelkoul H F J. antibiotic and immunomodulating properties of biochemical, 2014,2005(2):63.), anticancer (Paterson R M. Cordyceps-A biochemical and anticancer effects [ J.: 69, 2008-1469?). Polysaccharides in the extract are among the most abundant and important active ingredients, and have hypoglycemic activity (Zhang G, Huang Y, Bian Y, ethyl. hyperboglycemic activity of the fungi cordyces mileris, cordyces sitis, Tricholoma mongolicum, and Omphalia lapinos sensitive in streptozocin-induced diabetes rates [ J ]. Applied Microbiology and Biotechnology,2006,72(6): 1152-1156.). The solid strain of edible and medicinal fungi has long culture time, and long polysaccharide extraction period from fruiting body. The liquid fermentation can realize continuous production, large yield and short production period, and various substances with physiological activity, such as polysaccharide, polypeptide, alkaloid, terpenoid, vitamin, phytohormone and the like, are generated in the fermentation liquor to obtain the nutrient components similar to the sporocarp.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a preparation method of cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO. The method is stable and reliable and has strong practicability.
The invention also aims to provide the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO, which is prepared by the method.
The invention further aims to provide application of the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO.
The purpose of the invention is realized by the following technical scheme: a preparation method of cordyceps militaris polysaccharide with a function of inhibiting macrophage from secreting NO comprises the following steps:
(1) inoculating Cordyceps militaris strains in a liquid culture medium for fermentation culture, and performing solid-liquid separation to obtain Cordyceps militaris fermentation liquor and Cordyceps militaris mycelia;
(2) drying, crushing and sieving the cordyceps militaris mycelia obtained in the step (1) to obtain mycelium powder; then adding petroleum ether for degreasing treatment, and drying to obtain cordyceps militaris mycelium dry powder;
(3) adding the cordyceps militaris mycelium dry powder obtained in the step (2) into water, and performing hot water extraction at the temperature of 65-95 ℃ to obtain cordyceps militaris extract; then concentrating the cordyceps militaris leaching liquor to obtain cordyceps militaris leaching concentrated solution;
(4) removing protein from the cordyceps militaris leaching concentrated solution obtained in the step (3) and the cordyceps militaris fermentation liquor obtained in the step (1) by combining trypsin with a Sevage method respectively to obtain an intracellular crude polysaccharide extracting solution and an extracellular crude polysaccharide extracting solution after protein removal in sequence;
(5) respectively adding the intracellular crude polysaccharide extracting solution and the extracellular crude polysaccharide extracting solution obtained in the step (4) into an ethanol solution, standing, centrifuging, collecting precipitates, and respectively adding water for re-dissolving to obtain an intracellular crude polysaccharide solution and an extracellular polysaccharide solution; decolorizing, dialyzing, concentrating, and drying to obtain IPCM (intracellular polysaccharide extract of Cordyceps militaris) and EPCM (extracellular polysaccharide extract of Cordyceps militaris);
(6) performing DEAE-cellulose column chromatography purification on the IPCM and the EPCM obtained in the step (5), then dialyzing, and freeze-drying to obtain cordyceps militaris intracellular polysaccharide (IPCM-1) and cordyceps militaris extracellular polysaccharide (EPCM-1); the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO is obtained; wherein, DEAE-cellulose column chromatography adopts Tris-HCL buffer solution with pH 7.2 and containing 0.4mol/L NaCl to elute IPCM, and adopts Tris-HCL buffer solution with pH8.0 and containing 1mol/L NaCl to elute EPCM.
The Cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO is at least one of Cordyceps militaris intracellular polysaccharide (IPCM-1) and Cordyceps militaris extracellular polysaccharide (EPCM-1).
The formula of the liquid fermentation medium in the step (1) is as follows: sucrose 50g, KNO34g、KH2PO41g、MgSO4·7H2O1 g, vitamin B10.05g, pure water supplement to 1000mL, and pH value adjusted to 6.5.
The fermentation culture in the step (1) is preferably carried out by:
①, transferring the slant mother strain of cordyceps militaris after activated culture to a slant solid culture medium, and culturing for 8-15 days at 20-30 ℃ (preferably for 10 days at 27 ℃), so as to obtain the slant strain of cordyceps militaris;
and picking 4-6 bacterium blocks from the cordyceps militaris slant strain obtained in the step ①, inoculating the bacterium blocks into 150-200 mL of liquid fermentation medium, standing at room temperature for 24h, and carrying out light-proof constant-temperature oscillation culture for 6-7 d at the rotation speed of 150rpm at the temperature of 24 ℃ after the wound of the bacterium blocks is healed to obtain a first-stage seed solution.
thirdly, transferring the primary seed liquid obtained in the step ① into a 50L seed tank to culture a secondary seed liquid, wherein the culture temperature is 24 ℃, the rotating speed is 150rpm, the tank pressure is 0.05-0.07 MPa, and the ventilation volume is 1.5m3Culturing for 3 days, fermenting in 500L fermentation tank at the same temperature, rotation speed and pressure as seed tank and 12m ventilation3And h, culturing for 3-4 days.
the formula of the slant solid culture medium comprises 200g of peeled potato, 20g of glucose, 1g of peptone and (NH)4)2SO42g，MgSO4·7H2O 1g，KH2PO41g of agar and 20g of agar, adding distilled water to reach the constant volume of 1000mL, and adjusting the pH value to 6.5。
the culture medium for the first-stage seed liquid and the second-stage seed liquid culture and fermentation culture comprises 50g of sucrose and KNO34g、KH2PO41g、Mg SO4·7H2O1 g, vitamin B10.05g, pure water supplement to 1000mL, and pH value adjusted to 6.5.
The solid-liquid separation in the step (1) is separation by adopting a gauze filtering, centrifuging or suction filtering mode.
The gauze is preferably medical gauze; preferably 60-100 meshes of medical gauze.
The drying temperature in the step (2) is 50-70 ℃; preferably 60 deg.c.
Sieving in the step (2) is to pass through a sieve of 40-60 meshes; preferably 40 mesh.
The solid-to-liquid ratio of the mycelium powder to the petroleum ether in the step (2) is 1: 1-2 (g/mL); preferably 1:1 (g/mL).
The time of degreasing in the step (2) is 2-4 days; preferably 2 days.
The degreasing treatment in the step (2) is carried out for 1-3 times; preferably 2 times.
The feed-liquid ratio of the cordyceps militaris mycelium dry powder to water in the step (3) is 1: 20-40 (g/mL); preferably 1:25 (g/mL).
The temperature of the extraction in step (3) is preferably 75 ℃.
The extraction time in the step (3) is 1-3 h; preferably 2.5 h.
The extraction times in the step (3) are 1-3 times; preferably 2 times.
The concentration in the step (3) is performed by adopting a reduced pressure concentration mode.
The concentration temperature in the step (3) is 50-70 ℃; preferably 55-60 ℃; more preferably 60 deg.c.
The concentration in the step (3) is to concentrate the cordyceps militaris to 1/5-1/10 of the volume of the cordyceps militaris leaching liquor; preferably concentrating to 1/5 of the volume of Cordyceps militaris leaching solution.
The protein removal by adopting the trypsin combined Sevage method in the step (4) is preferably realized by the following steps:
(I) respectively adjusting the pH of the cordyceps militaris leaching concentrated solution obtained in the step (3) and the cordyceps militaris fermentation liquor obtained in the step (1) to 8.0, then respectively adding a trypsin solution, uniformly mixing, oscillating at 37 ℃ and 120rpm for 30-60 min (preferably 30min), then inactivating enzymes in a water bath, and cooling to room temperature to obtain a cordyceps militaris intracellular leaching liquor and a cordyceps militaris extracellular leaching liquor after enzymolysis;
(II) adding the enzymolyzed Cordyceps militaris intracellular leaching liquor and Cordyceps militaris extracellular leaching liquor obtained in step (I) into a chloroform-n-butanol mixed liquor respectively, oscillating vigorously for 30-40 min (preferably 30min), centrifuging, removing a protein layer and an organic solution, and collecting a supernatant;
(III) repeating the step (II) for 3-5 times until no protein layer appears any more, and obtaining the intracellular crude polysaccharide extracting solution and the extracellular crude polysaccharide extracting solution after protein removal.
The enzyme deactivation conditions in the step (I) are as follows: inactivating enzyme for 10-15 min at 100 ℃; preferably: inactivating enzyme at 100 deg.C for 10 min.
The concentration of the trypsin solution in the step (I) is 2-5% by mass volume ratio; preferably 2%.
The volume ratio of the trypsin solution to the concentrated cordyceps militaris leaching liquor in the step (I) is 1: 20-30; preferably 1: 20.
the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution in the step (II) is 5: 1.
the volume ratio of the chloroform-n-butanol mixed solution in the step (II) to the cordyceps militaris extracellular leaching liquor after enzymolysis (or cordyceps militaris intracellular leaching liquor after enzymolysis) is 1: 0.2 to 0.25; preferably 1: 0.2.
the conditions of centrifugation described in step (II) are: centrifuging at 5000-8000 rpm for 10-15 min; preferably: centrifuge at 8000rpm for 10 min.
The volume ratio of the ethanol solution in the step (5) to the intracellular crude polysaccharide extracting solution (or extracellular crude polysaccharide extracting solution) except the protein is 2-5: 1; preferably 4-5: 1; more preferably 4: 1.
The ethanol solution in the step (5) is an ethanol solution with a volume fraction of 95%.
The standing condition in the step (5) is as follows: standing for 24-48 h at 0-4 ℃; preferably: standing at 4 ℃ for 48 h.
The centrifugation conditions in the step (5) are as follows: centrifuging at 5000-8000 rpm for 10-15 min; preferably: centrifuge at 8000rpm for 10 min.
The decolorization in the step (5) is performed by adopting H2O2Decoloring the solution; preferably, the mass fraction of H is 30 percent2O2The solution is decolorized.
Said H2O2The dosage of the solution is 1/30-1/50 of the volume of the polysaccharide solution; preferably 1/50 of the volume of the polysaccharide solution.
The concentration of the intracellular crude polysaccharide solution in the step (5) is 15-30 mg/mL; preferably 20 to 30 mg/mL.
The concentration of the extracellular polysaccharide solution in the step (5) is 15-30 mg/mL; preferably 20 to 30 mg/mL.
The decoloring conditions in the step (5) are as follows: and (4) preserving heat for 1-2 h in a 50 ℃ water bath.
The dialysis in the step (5) is carried out by adopting a dialysis bag with the molecular weight cutoff of 6000-8000 Da; preferably, a dialysis bag with the molecular weight cutoff of 6000-8000 Da is adopted for dialysis for 72h, and deionized water is replaced every 8 h.
Concentrating the cordyceps militaris polysaccharide extract to 1/3-1/5 of the cordyceps militaris polysaccharide extract in volume in the step (5); preferably concentrating to 1/3 of the volume of the Cordyceps militaris polysaccharide extractive solution.
The DEAE-cellulose column chromatography described in the step (6) is preferably carried out by:
s1, column packing: adding 1/3 column volume of deionized water into the chromatographic column, opening a liquid outlet at the lower end, and pouring DEAE-cellulose filler into the chromatographic column to naturally settle in the chromatographic column, wherein the volume of the filler is 2/3 of the column volume;
s2, balancing: pumping the Tris-HCl buffer solution into the chromatographic column by using a constant flow pump, and opening a liquid outlet at the lower end until the pH value of an effluent liquid is the same as that of the Tris-HCl buffer solution;
s3, loading and elution:
s31, dissolving IPCM in Tris-HCL with the pH value of 8.0, centrifuging, and taking supernatant to obtain IPCM solution; adding IPCM solution into chromatographic column, eluting with Tris-HCl buffer solution (sodium chloride dissolved in Tris-HCl buffer solution) with pH of 7.2 and containing 0.4mol/L NaCl, and collecting intracellular polysaccharide of Cordyceps militaris; measuring total sugar content of intracellular polysaccharide of Cordyceps militaris by phenol-sulfuric acid method, mixing single absorption peak samples, dialyzing, concentrating, and freeze drying to obtain intracellular polysaccharide (IPCM-1) of Cordyceps militaris;
s32, dissolving the EPCM in a Tris-HCL buffer solution with the pH value of 8.2, centrifuging, and taking a supernatant to obtain an EPCM solution; then adding the EPCM solution into a chromatographic column, eluting with a Tris-HCl buffer solution with the pH value of 8.0 and containing 1mol/L NaCl, and collecting cordyceps militaris extracellular polysaccharide; measuring total sugar content of intracellular polysaccharide of Cordyceps militaris by phenol-sulfuric acid method, mixing single absorption peak samples, dialyzing, concentrating, and freeze drying to obtain extracellular polysaccharide (EPCM-1) of Cordyceps militaris.
The specification of the chromatography column described in step S1 is: 3.0cm × 80 cm.
The pH value of the Tris-HCl buffer solution in the step S2 is 7.0-7.6; preferably 7.0.
The flow rate of the effluent described in step S2 was 1 mL/min.
The concentration of the IPCM solution in the step S31 is 10-15 mg/mL; preferably 10 mg/mL.
The conditions of centrifugation in step S31 and step S32 are: centrifuging at 5000-8000 rpm for 10 min; preferably: centrifuge at 8000rpm for 10 min.
The concentration of the EPCM solution in the step S32 is 10-15 mg/mL; preferably 10 mg/mL.
The flow rate of elution described in step S31 and step S32 was 1 mL/min.
The dialysis in the step (6) is carried out by adopting a dialysis bag with the molecular weight cutoff of 6000-8000 Da; preferably, a dialysis bag with the molecular weight cutoff of 6000-8000 Da is adopted for dialysis for 72 h.
The preparation method of the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO further comprises the step of purifying the obtained cordyceps militaris intracellular polysaccharide (IPCM-1) and cordyceps militaris extracellular polysaccharide (EPCM-1) by using a cross-linked Sephadex G-100 gel column after the step (6); the method specifically comprises the following steps:
(i) adding 1/3 column volume of deionized water into the chromatographic column, opening a liquid outlet at the lower end, filling the swelled crosslinked Sephadex G-100 gel into the chromatographic column, naturally settling the gel in the chromatographic column, and balancing the gel with distilled water for 2-3 hours after filling;
(ii) dissolving Cordyceps militaris intracellular polysaccharide (IPCM-1) and Cordyceps militaris extracellular polysaccharide (EPCM-1) in water respectively to obtain IPCM-1 solution and EPCM-1 solution; and then respectively adding the IPCM-1 solution and the EPCM-1 solution into a chromatographic column by using a constant flow pump, eluting by using ultrapure water, controlling the flow rate at 1mL/min, measuring the total sugar content by using a phenol-sulfuric acid method, combining single absorption peaks, concentrating and carrying out vacuum freeze drying treatment to obtain purified cordyceps militaris intracellular polysaccharide (IPCM-2) and cordyceps militaris extracellular polysaccharide (EPCM-2).
The concentration of the IPCM-1 solution in the step (ii) is 10-15 mg/mL; preferably 10 mg/mL.
The concentration of the EPCM-1 solution in the step (ii) is 10-15 mg/mL; preferably 10 mg/mL.
The number average molecular weight (Mn) of the intracellular polysaccharide (IPCM-2) of the cordyceps militaris in the step (ii) is 1.61 multiplied by 104Da, weight average molecular weight (Mw) of 3.25X 104Da; the number average molecular weight (Mn) of extracellular polysaccharide (EPCM-2) of Cordyceps militaris is 1.34 × 104Da, weight average molecular weight (Mw) of 2.00X 104Da。
Cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO is prepared by any one of the methods.
The cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO is applied to preparation of anti-inflammatory drugs or health-care foods, and has a good effect of inhibiting nitric oxide secretion in macrophages.
The effective concentration of the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO is 20-320 mug/mL; preferably 20 to 320. mu.g/mL.
The cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO is applied to preparation of a medicine for inhibiting macrophage from secreting NO; wherein said NO is secreted by Lipopolysaccharide (LPS) stimulating macrophages.
The application is realized by any one of the following modes:
(A) simultaneously adding cordyceps militaris polysaccharide and lipopolysaccharide into macrophages for cell culture so as to inhibit the macrophages from secreting NO;
(B) adding cordyceps militaris polysaccharide into macrophages, culturing for 6-24 h, replacing with a culture medium without polysaccharide, and adding LPS to stimulate cells for 12h to inhibit NO secretion of the macrophages.
The effective concentration of the cordyceps militaris polysaccharide in the mode (A) is 20-320 mu g/mL; preferably 20-160 mu g/mL; preferably 20 to 160. mu.g/mL.
The cordyceps militaris polysaccharide in the mode (A) is preferably cordyceps militaris exopolysaccharide (EPCM-2).
The culture time in the mode (A) is 6-24 h; preferably 12 h.
The concentration of Lipopolysaccharide (LPS) described in the modes (A) and (B) was 1. mu.g/ml.
The effective concentration of the cordyceps militaris polysaccharide in the mode (B) is 20-160 mug/mL; preferably 160. mu.g/mL.
The cordyceps militaris polysaccharide in the mode (B) is preferably cordyceps militaris intracellular polysaccharide (IPCM-2).
The time for the cultivation in the mode (B) is preferably 6 hours.
Compared with the prior art, the invention has the following advantages and effects:
1. the cordyceps militaris extract can be prepared by adopting a fermentation tank, mycelia and fermentation liquor are collected to extract cordyceps militaris intracellular polysaccharide and cordyceps militaris extracellular polysaccharide, the preparation method is simple and quick, the used reagent is mild, the operation is safe, no toxicity is caused to human bodies, the conditions are easy to control, the practicability is high, and batch production can be carried out.
2. The chemical culture medium used in the invention, namely the culture medium taking sucrose and potassium nitrate as carbon-nitrogen sources, has the advantages of accurate components, strong repeatability, good growth of mycelium, easy separation and purification, and negligible influence of the culture medium on the mycelium in polysaccharide determination.
3. In the invention, the DEAE-cellulose column chromatography and the sephadex G-100 column are combined to purify the polysaccharide, the purification effect is good, and the used reagent is simple and easy to prepare and has high repeatability.
4. The intracellular polysaccharide and the extracellular polysaccharide of the cordyceps militaris obtained by the invention have good water solubility, can inhibit macrophages from secreting NO, and have good anti-inflammatory effect.
Drawings
FIG. 1 is a standard curve diagram of different molecular weight dextran standards GPC.
FIG. 2 is a GPC chromatogram of IPCM-2.
FIG. 3 is a GPC chromatogram of EPCM-2.
FIG. 4 is a graph showing the effect of Cordyceps militaris polysaccharides on macrophage survival (compare with control group without polysaccharide: # P <0.01, # P < 0.05).
FIG. 5 is a graph showing the effect of Cordyceps militaris polysaccharides on NO secretion by macrophages (compare to LPS-only treated group: # P <0.01, # P < 0.05).
FIG. 6 is a graph showing the effect of Cordyceps militaris polysaccharides on NO secretion from RAW264.7 cells over time with LPS (compared to blank control: # P <0.01# P < 0.05; compared to LPS-only treated group: # P <0.01, # P < 0.05).
FIG. 7 is a graph showing the effect of polysaccharide pre-action of Cordyceps militaris on the secretion of NO by LPS-induced RAW264.7 cells (compared to blank control: # P <0.01, # P < 0.05; compared to LPS-only treated group: # P <0.01, # P < 0.05).
FIG. 8 is a graph showing the effect of cordyceps militaris polysaccharide pre-action time on NO secretion by LPS-induced RAW264.7 cells (compared to blank control.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The following examples are given without reference to specific experimental conditions, and are generally in accordance with conventional experimental conditions. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
The Cordyceps militaris (C.militaris) strains referred to in the examples were provided by the national institute of bioscience of south China university (Ma L, Zhang S, et al. Hyperuricemic Actions of Exopolyscopide Produced by City millerits militaris in Potasidum Oxonate-Induced Hyperuricemic Mice. Current microbiology,69(6):852-857, 2014.).
The media formulations referred to in the examples are as follows:
the formula of the slant solid culture medium is as follows: potato (peeled) 200g, glucose 20g, peptone 1g, (NH)4)2SO42g，MgSO4·7H2O 1g，KH2PO41g of agar and 20g of agar, adding distilled water to a constant volume of 1000mL, and adjusting the pH value to 6.5;
the formula of the liquid fermentation medium is as follows: sucrose 50g, KNO34g、KH2PO41g、Mg SO4·7H2O1 g, vitamin B10.05g, 1000mL of water, pH 6.5.
Example 1
(1) Slant or plate strain culture, liquid shaking culture and liquid submerged fermentation culture are carried out on cordyceps militaris (C.militaris) to obtain mycelium, and the specific method comprises the following steps:
①, slant or plate strain culture, namely inoculating activated cordyceps militaris slant mother strain hypha blocks into a new slant solid culture medium, and culturing for 10 days at 27 ℃ for later use;
②, first-stage liquid strain culture, namely putting 200mL of liquid fermentation medium into a 500mL triangular flask, sterilizing at 121 ℃ for 20min, inoculating cordyceps militaris mother strain mycelia with the size of 4-6 soybeans, standing at room temperature for 24h, and after the wounds of the mycelia heal, carrying out light-proof constant-temperature oscillation culture at 24 ℃ and 150rpm for 6 days to obtain first-stage seed liquid;
thirdly, secondary liquid strain culture, namely 35L of liquid fermentation culture medium is filled into a 50L seed tank, sterilization is carried out for 20min at 125 ℃, the cultured primary seed liquid is inoculated after cooling, the culture temperature is 24 ℃, the rotating speed is 150rpm, the tank pressure is 0.05-0.07 MPa, and the ventilation volume is 1.5m3H, culturing for 3 days, sampling and observing to find that the seed liquid is clear and bright, the mycelium is rich and the size is uniform, and obtaining a second-stage seed liquid；
fourthly, liquid submerged fermentation culture, namely transferring the cultured secondary seed liquid into a 500L fermentation tank for culture, wherein the temperature, the rotating speed and the tank pressure of the initial culture condition are the same as those of the seed tank, and the ventilation volume is about 12m3H, culturing for 4 d;
(2) filtering the fermentation product by medical gauze (60-100 meshes) to obtain fermentation liquor and mycelia of cordyceps militaris fermentation; then putting the mycelium into an oven to dry to constant weight for later use; wherein the drying temperature is set to 60 ℃.
(3) Degreasing: and drying the mycelium to constant weight, crushing the mycelium by a crusher, sieving the mycelium by a 40-mesh sieve, and collecting mycelium powder of more than 40 meshes. Placing the mycelium powder in a triangular flask, uniformly stirring the mycelium powder and petroleum ether according to the solid-to-liquid ratio of 1:1(g/mL), placing on a shaking table, shaking for 48h, centrifuging to retain precipitate, and repeating the steps once. The obtained precipitate is dried at 60 ℃ and placed in a drying box for standby.
(4) Hot water leaching: extracting the degreased and dried cordyceps militaris mycelia in the step (3) with hot water, and collecting an extract liquid; wherein: extracting at 75 deg.C for 2 times for 2.5h at a material-to-liquid ratio of 1:25(g: ml);
(5) concentration: respectively carrying out reduced pressure concentration on the leaching liquor collected in the step (4) and the fermentation liquor obtained in the step (2) in a constant-temperature water bath kettle at the temperature of 60 ℃ to 1/5 of the original volume by using a rotary evaporator to obtain a cordyceps militaris leaching concentrated solution and a fermentation concentrated solution;
(6) trypsin and sevage method protein removal, centrifugation: 2.0g of trypsin is respectively weighed and dissolved in 100mL of distilled water to obtain 2 parts of the same trypsin solution; regulating the pH value of 1000mL of the cordyceps militaris extraction concentrated solution and the fermentation concentrated solution to 8.0, respectively adding a trypsin solution into the cordyceps militaris extraction concentrated solution and the fermentation concentrated solution, preheating the mixture in a 37 ℃ water bath kettle for 10min, respectively and fully mixing an enzyme solution and the two concentrated solutions, oscillating the mixture at 120rpm for 30min, then putting the mixture in a 100 ℃ water bath kettle for enzyme deactivation for 10min, and cooling the mixture to room temperature; a 0.2-fold volume of chloroform-n-butanol mixture (chloroform: n-butanol (V: V) ═ 5:1) was added, and the mixture was vigorously shaken for 30 min. Centrifuging at 8000rpm for 10min, discarding protein layer and organic solution, recovering supernatant, repeating the process for 2 times until no protein layer appears, and respectively obtaining crude polysaccharide extractive solution after protein removal;
(7) adding 4 times volume of 95% ethanol into the two polysaccharide extracts after protein removal, placing the two polysaccharide extracts in a refrigerator at 4 ℃ for cold storage for 48h, and centrifuging (at 5000rpm for 10min) to obtain precipitates after crude polysaccharide is separated out;
② dissolving the precipitate (polysaccharide solution concentration is 20mg/mL) with a small amount of distilled water respectively, adjusting the pH of the polysaccharide solution to 8.0, then dripping 30 percent of H by mass fraction2O2(2 mL of 30% H was added dropwise to 100mL of polysaccharide solution2O2Solution), keeping the temperature of the solution in water bath for 2 hours at 50 ℃, and performing decoloration treatment on the solution;
filling the decolorized polysaccharide solution into dialysis bags with the molecular weight cutoff of 6000-8000 Da respectively, dialyzing for about 72 hours with distilled water, and replacing the distilled water once every 8 hours;
and fourthly, respectively concentrating the dialyzed solution (to 1/3 of the original volume), and performing vacuum freeze drying to obtain the cordyceps militaris intracellular polysaccharide extract (IPCM) and the cordyceps militaris extracellular polysaccharide Extract (EPCM).
The cordyceps militaris intracellular polysaccharide and extracellular polysaccharide extracts respectively contain 54.37 percent of total sugar and 58.26 percent of total sugar by measuring by a phenol-sulfuric acid method; respectively containing reducing sugar 9.35% and reducing sugar 7.21% by DNS colorimetric method; the protein content is respectively 0.22% and 0.96% (the contents are mass fractions) determined by Coomassie Brilliant blue method.
Example 2
(1) Slant or plate strain culture, liquid shaking culture and liquid submerged fermentation culture are carried out on cordyceps militaris (C.militaris) to obtain mycelium, and the specific method comprises the following steps:
①, slant or plate strain culture, namely inoculating activated cordyceps militaris slant mother strain hypha blocks into a new slant solid culture medium, and culturing for 10 days at 27 ℃ for later use;
②, first-stage liquid strain culture, namely putting 200mL of liquid fermentation medium into a 500mL triangular flask, sterilizing at 121 ℃ for 20min, inoculating cordyceps militaris mother strain mycelia with the size of 4-6 soybeans, standing at room temperature for 24h, and after the wounds of the mycelia heal, carrying out light-proof constant-temperature oscillation culture at 24 ℃ and the rotation speed of 150rpm for 6 days to obtain first-stage seed liquid;
thirdly, secondary liquid strain culture, namely 35L of liquid fermentation culture medium is filled into a 50L seed tank, sterilization is carried out for 20min at 125 ℃, the cultured primary seed liquid is inoculated after cooling, the culture temperature is 24 ℃, the rotating speed is 150rpm, the tank pressure is 0.05-0.07 MPa, and the ventilation volume is 1.5m3Culturing for 3 days, and sampling and observing to obtain clear and transparent seed liquid, rich mycelia and uniform size to obtain secondary seed liquid;
fourthly, liquid submerged fermentation culture, namely transferring the cultured secondary seed liquid into a 500L fermentation tank for culture, wherein the temperature, the rotating speed and the tank pressure of the initial culture condition are the same as those of the seed tank, and the ventilation volume is about 12m3H, culturing for 4 d;
(2) filtering the fermentation product by medical gauze (60-100 meshes) to obtain fermentation liquor and mycelia of cordyceps militaris fermentation; then putting the mycelium into an oven to dry to constant weight for later use; wherein the drying temperature is set to 60 ℃.
(3) Degreasing: and drying the mycelium to constant weight, crushing the mycelium by a crusher, sieving the mycelium by a 40-mesh sieve, and collecting mycelium powder of more than 40 meshes. Placing the mycelium powder in a triangular flask, uniformly stirring the mycelium powder and petroleum ether according to the solid-to-liquid ratio of 1:1(g/mL), placing on a shaking table, shaking for 48h, centrifuging to retain precipitate, and repeating the steps once. The obtained precipitate is dried at 60 ℃ and placed in a drying box for standby.
(4) Hot water leaching: extracting the degreased and dried cordyceps militaris mycelia in the step (3) with hot water, and collecting an extract liquid; wherein: extracting at 75 deg.C for 2 times for 2.5h at a material-to-liquid ratio of 1:25(g: ml);
(5) concentration: respectively carrying out reduced pressure concentration on the leaching liquor collected in the step (4) and the fermentation liquor obtained in the step (2) in a constant-temperature water bath kettle at the temperature of 60 ℃ to 1/5 of the original volume by using a rotary evaporator to obtain a cordyceps militaris leaching concentrated solution and a fermentation concentrated solution;
(6) deproteinization by trypsin and sevage method, centrifugation: respectively weighing 2.0g of trypsin and dissolving in 100mL of distilled water to obtain 2 parts of trypsin solution with the mass fraction of 2%; respectively adjusting the pH value of 1000mL of the cordyceps militaris extraction concentrated solution and the fermentation concentrated solution to 8.0, respectively adding a trypsin solution, then putting the mixture into a 37 ℃ water bath pot for preheating for 10min, respectively and fully mixing the enzyme solution, the extraction concentrated solution and the fermentation concentrated solution, oscillating at 120rpm for 30min, then putting the mixture into a 100 ℃ water bath pot for inactivating the enzyme for 10min, and cooling to room temperature; a 0.2-fold volume of chloroform-n-butanol mixture (chloroform: n-butanol (V: V) ═ 5:1) was added, and the mixture was vigorously shaken for 30 min. Centrifuging at 8000rpm for 10min, discarding protein layer and organic solution, recovering supernatant, repeating the process for 2 times until no protein layer appears, and respectively obtaining crude polysaccharide extractive solution after protein removal;
(7) adding 4 times volume of 95% ethanol into the two polysaccharide extracts after deproteinization, placing in a refrigerator at 4 ℃ for cold storage for 48h, and centrifuging (centrifuging at 5000rpm for 10min) to obtain precipitate after crude polysaccharide is separated out;
② dissolving the precipitate (polysaccharide solution concentration is 30mg/mL) with a small amount of distilled water respectively, adjusting the pH value of the polysaccharide solution to 8.0, then dripping 30 percent of H by mass fraction2O2(2 mL of 30% H was added dropwise to 100mL of polysaccharide solution2O2Solution), keeping the temperature of the solution in water bath for 2 hours at 50 ℃, and performing decoloration treatment on the solution;
filling the decolorized polysaccharide solution into dialysis bags with the molecular weight cutoff of 6000-8000 Da respectively, dialyzing for about 72 hours with distilled water, and replacing the distilled water once every 8 hours;
and fourthly, respectively concentrating the solution after dialysis (concentrating to 1/3 of the original volume), and performing vacuum freeze drying to obtain the intracellular polysaccharide extract (IPCM) and the extracellular polysaccharide Extract (EPCM) of the cordyceps militaris.
(8) Purifying the polysaccharide of the obtained cordyceps militaris intracellular polysaccharide and extracellular polysaccharide extract by using a DEAE-cellulose ion exchange resin column, and specifically comprising the following steps of:
(ii) pretreatment of DEAE-cellulose
100g of DEAE-cellulose (Whatman, UK) was immersed in distilled water for 24 hours, and the excess fraction was removed by flotation, and the precipitate fraction was collected. Adding NaOH solution into per gram of the mixture at a ratio of 15mL to 0.5mol/L NaOH, soaking for 30min, intermittently stirring for multiple times, loading into a chromatographic column (3.0cm × 80cm, Ware glass instruments, Guangzhou), washing with distilled water to neutrality, and pouring out. Soaking in 0.5mol/L HCl solution for 30min, intermittently stirring for several times, loading into chromatographic column, washing with distilled water to neutrality, and pouring out. And then, continuously soaking the column in 0.5mol/L NaOH solution for 30min, filling the column, and finally washing the column with distilled water until the pH value is neutral for later use.
② DEAE-cellulose purification process of polysaccharide
Column assembling: after the chromatographic column is cleaned, the chromatographic column is vertically fixed on an iron support, deionized water with about 1/3 of the column volume is added into the chromatographic column, a lower liquid outlet is opened, DEAE-cellulose resin is slowly poured into the chromatographic column to be naturally settled in the chromatographic column, the volume of the resin is about 2/3 of the column volume, and the column filling is stopped. The liquid inlet at the upper end of the chromatographic column is connected with a constant flow pump.
Balancing: pumping Tris-HCL buffer solution (pH is 7.0) into the chromatographic column by using a constant flow pump, opening a liquid outlet at the lower end of the chromatographic column, keeping the flow rate of the equilibrium buffer solution at about 1mL/min, and balancing the chromatographic column when the pH value of effluent liquid at a lower liquid outlet is the same as that of the Tris-HCL buffer solution.
Loading and eluting: 0.1g IPCM was dissolved in 10mL Tris-HCl pH8.0, 0.1g EPCM was dissolved in 10mL Tris-HCl buffer pH 8.2, then centrifuged at 8000rpm for 10min, the supernatant was taken and slowly added to the equilibrated column using a constant flow pump. Washing off unadsorbed substances by using a buffer solution, eluting IPCM by using a Tris-HCl buffer solution with the pH value of 7.2 and containing 0.4mol/L NaCl, eluting EPCM by using a Tris-HCl buffer solution with the pH value of 8.0 and containing 1mol/L NaCl, controlling the flow rate at 1mL/min, setting the collection time of a partial collector (the collection time is 10 minutes per tube), and collecting. And (3) determining the total sugar content by a phenol-sulfuric acid method, combining samples with single absorption peaks, dialyzing for 72h by using distilled water (the cut-off molecular weight of a dialysis bag is 6000-8000 Da), and replacing the distilled water once every 8 h. After dialysis, the dialyzate is concentrated and vacuum freeze-dried to obtain purified intracellular polysaccharide (IPCM-1) and extracellular polysaccharide (EPCM-1).
(9) Polysaccharide purification by cross-linked Sephadex G-100 gel column
pretreatment of crosslinked Sephadex G-100 gel
Cross-linked Sephadex G-100 gel (Whatman, UK) was treated by the room temperature swelling method: firstly weighing Sephadex G-10050G, adding 100mL of distilled water, standing at room temperature for 24h to fully swell, and removing the superfine part by a flotation method for later use.
② Sephadex G-100 purification of polysaccharide
Column assembling: the chromatographic column is vertically arranged on an iron support, about 1/3 column volume of distilled water is added firstly, then the swelled cross-linked Sephadex G-100 gel is continuously and slowly filled (2/3 column volume after gel sedimentation) while being stirred uniformly, so that the gel is naturally settled in the chromatographic column, and a lower liquid outlet is opened at the same time. The gel after column packing must be uniform without bubbles or significant streaking. Otherwise, the gel must be poured out and refilled. After the sample is filled, the sample can be added after the sample is balanced for 2-3 hours by using distilled water.
Loading and eluting: 0.1g of IPCM-1 and EPCM-1 are respectively dissolved in 10mL of ultrapure water to obtain IPCM-1 solution and EPCM-1 solution; then slowly adding the IPCM-1 solution and the EPCM-1 solution into the balanced chromatographic column respectively by using a constant flow pump. When in elution, ultrapure water is used for elution, and the elution is continuously carried out, so that a certain liquid layer is kept at the upper end of the gel column, and the liquid on the surface of the gel column is prevented from flowing dry; the flow rate was controlled at 1mL/min, and the collection time of the fraction collector (10 minutes per tube) was set for collection. Measuring total sugar content by phenol-sulfuric acid method, combining single absorption peaks, concentrating, and vacuum freeze drying to obtain purified intracellular polysaccharide (IPCM-2) and extracellular polysaccharide (EPCM-2).
The polysaccharide content of the cordyceps militaris intracellular polysaccharide extracts (IPCM-1 and IPCM-2) is respectively determined by a sulphuric acid phenol method as follows: 71.38% and 91.63% (mass fraction); the polysaccharide content of the cordyceps militaris extracellular polysaccharide extracts (EPCM-1 and EPCM-2) measured by a sulfuric acid phenol method is respectively as follows: 60.93 percent and 91.68 percent (mass fraction).
(10) Determination of polysaccharide molecular weight
The relative molecular weights of IPCM-2 and EPCM-2 were determined by GPC.
GPC conditions are TSK-GEL G3000SW XL (TOSOH corporation) as a chromatographic column of 300mm × 718mm at a column temperature of 35 ℃ and 0.05mol/L NaH as a mobile phase2PO4-Na2HPO4Buffer (pH 6.7, 0.05% (w/v) NaN3) (ii) a Volume flow rate: 0.8 mL/min; refractive index detector, constant temperature35 ℃; the amount of the sample was 20. mu.L.
establishment of GPC calibration curve with known relative molecular mass of 5800, 1.22 × 104、2.37×104、4.8×104、1.0×105、1.86×105、3.8×105And 8.53X 105Da dextran standard (south China Standard substance Net) mixture, 0.05mol/L NaH2PO4-Na2HPO4Buffer (pH 6.7, 0.05% (w/v) NaN added3) Dissolved, filtered through a 0.22 μm filter and analyzed by GPC. Taking the retention time of the polysaccharide standard sample as an abscissa and the logarithm of the polysaccharide relative to the molecular mass as an ordinate to prepare a universal calibration curve of GPC.
analysis of samples 20mg of IPCM-2 and EPCM-2 samples were sampled at 4ml of 0.05mol/L of NaH2PO4-Na2HPO4Buffer (pH 6.7, 0.05% NaN3) Dissolved, filtered through a 0.22 μm filter and analyzed by GPC. And (4) calculating the average relative molecular mass and the distribution of polysaccharide peaks on the calibration curve according to the peak emergence time of the sample.
The linear relationship between the molecular weight and the elution volume can be obtained according to the GPC chart of the dextran standard with different molecular weights by detecting the dextran standard with different molecular weights through GPC, and is shown in FIG. 1. IPCM-2 and EPCM-2 were detected by GPC, and the results are shown in FIGS. 2 and 3. Analysis according to linear relationship and GPC software gave: the number average molecular weight (Mn) of IPCM-2 is 1.61X 104Da, weight average molecular weight (Mw) of 3.25X 104Da; EPCM-2 number average molecular weight (Mn) of 1.34X 104Da, weight average molecular weight (Mw) of 2.00X 104Da. Compared with other cordyceps militaris polysaccharide molecular weight researches, the molecular weight of IPCM-2 and EPCM-2 obtained by the experiment is slightly larger than other research results.
Example 3 experiment of Cordyceps militaris polysaccharide extracts IPCM-2 and EPCM-2 (prepared in example 2) for inhibiting NO secretion of macrophage
(1) MTT method for determining influence of cordyceps militaris polysaccharide on survival rate of macrophages
Observing the influence of Cordyceps militaris polysaccharide on the activity of RAW264.7 macrophage (animal experiment center of Zhongshan university) by using cell proliferation effect, and detecting fine particles by MTT methodCell proliferation with a cell density of 2X 106The culture medium for experiments was DMEM high-glucose culture medium (GIBCO, USA) containing 10% (v/v) fetal bovine serum (ilex purpurea, China). After the adherence, the cells were cultured with purified intracellular polysaccharide (IPCM-2) and extracellular polysaccharide (EPCM-2) (0, 20, 80, 320, 1280, 2000. mu.g/mL) for 24h, treated with 5mg/mL MTT for 24h, and cultured for 4 h. The medium was converted to 150. mu.L of DMSO (dimethyl sulfoxide), and after 30min of shaking, the absorbance at 490nm of each well was measured using a microplate reader (A490).
(2) Determination of NO content
RAW264.7 macrophages were seeded into 96-well culture plates for overnight culture (cell density 2X 10)6one/mL), then 0, 20, 40, 80, 160 μ g/mL cordyceps militaris polysaccharide (IPCM-2, EPCM-2) and 1 μ g/mL LPS (lipopolysaccharide) (SIGMA, USA) act on RAW264.7 macrophage for 24h at the same time, a blank control group uses a DMEM culture medium without cordyceps militaris polysaccharide and LPS to culture cells for 24h, an LPS treatment group uses a culture medium only containing 1 μ g/mL LPS to culture cells for 24h, and the content of NO in the culture medium supernatant is detected (by using a total nitric oxide detection kit (Biyun, China, the same below).
(3) Effect of different action time and different action method on NO secretion of RAW264.7 cells
influence of time of simultaneous action of cordyceps militaris polysaccharide and LPS on secretion of NO by LPS-induced macrophages
RAW264.7 macrophages were seeded into 96-well culture plates for overnight culture (cell density 2X 10)6one/mL), then 40 mu g/mL cordyceps militaris polysaccharide (IPCM-2 and EPCM-2) and 1 mu g/mL LPS are used by an experimental group to simultaneously act on RAW264.7 cells at different times, a blank control group is used for culturing the cells at different times by using a culture medium without the cordyceps militaris polysaccharide and the LPS, a positive control group (namely LPS group) is used for only stimulating the cells by 1 mu g/mL LPS for different times, and the content of NO in the culture medium supernatant of each group is checked at 6h, 12h, 18h and 24h respectively.
② influence of cordyceps militaris polysaccharide pre-action on LPS-induced NO secretion
RAW264.7 macrophages were seeded into 96-well culture plates for overnight culture (cell density 2X 10)6one/mL), then 0 for experiment20, 40, 80, 160 mu g/mL of cordyceps militaris polysaccharide (IPCM-2, EPCM-2) acts on RAW264.7 cells for 24 hours, then a new culture medium without polysaccharide is used, 1 mu g/mL of LPS is used for stimulating the cells for 12 hours, and the content of NO in cell culture supernatant is detected.
③ Effect of Cordyceps militaris polysaccharide pre-action time on LPS-induced RAW264.7 cell NO secretion
RAW264.7 macrophages were seeded into 96-well culture plates for overnight culture (cell density 2X 10)6one/mL), pre-acting the RAW264.7 cells of the experimental group for 6 hours, 12 hours and 24 hours respectively through 40 mu g/mL cordyceps militaris polysaccharide (IPCM-2 and EPCM-2), and then acting the cells for 12 hours through 1 mu g/mL LPS, and detecting the content of NO in the cell culture supernatant.
(4) The experimental results are as follows: the experimental results are shown in FIGS. 4 to 8.
To avoid cell death from cordyceps militaris polysaccharide treatment, the safe concentration range of the polysaccharide was first determined. FIG. 4 is a graph of the effect of Cordyceps militaris polysaccharides on macrophage survival. As can be seen from FIG. 4, the concentration of intracellular and extracellular polysaccharide of Cordyceps militaris in the range of 0-2000 μ g/mL has no killing power on the growth of RAW264.7 cells, and when the concentration is 320-2000 μ g/mL, the cells are remarkably proliferated (P <0.01), and the total number of cells shows an increasing trend. However, the cell density can be increased due to the proliferation of cells, the difference between the cell number of the culture medium added with the cordyceps militaris polysaccharide and the culture medium without the cordyceps militaris in the experimental process can influence the accuracy of the experimental result, and the cell activity is remarkably increased (P is less than 0.01) only after the cordyceps militaris polysaccharide concentration reaches 320 mu g/mL, so that the experimental concentration range of the cordyceps militaris polysaccharide acting on RAW264.7 cells is preferably selected to be 0-320 mu g/mL.
The effect of cordyceps militaris polysaccharide on NO secretion by macrophages is shown in fig. 5. RAW264.7 alone at 1. mu.g/ml LPS induced very significant NO secretion in cells compared to the blank control group (P < 0.01). Within the concentration range of 0-160 mu g/ml, the simultaneous action of intracellular polysaccharide and extracellular polysaccharide of cordyceps militaris and LPS can remarkably inhibit LPS induced RAW264.7 cells from secreting NO (P <0.01) and has NO remarkable difference (P >0.05) from a blank control group. Wherein the inhibition rates of 20, 40, 80 and 160 mu g/ml intracellular polysaccharide on NO secretion of macrophages are 56.04%, 62.34%, 56.05% and 53.65% respectively. And the inhibition rates of 20, 40, 80 and 160 mu g/ml exopolysaccharide on NO secretion of macrophages are 68.01%, 68.01%, 66.75% and 51.00% respectively. Experimental results show that the cordyceps militaris polysaccharide can effectively inhibit NO secretion, and the inhibition effect of extracellular polysaccharide (EPCM-2) is generally better than that of intracellular polysaccharide (IPCM-2) within the same concentration range.
The influence of the simultaneous action of the cordyceps militaris polysaccharide and the LPS on NO secretion of RAW264.7 cells in different time periods is shown in FIG. 6. As can be seen from FIG. 6, the NO secretion of each treatment group is sharply increased within 6h to 12h, and basically stabilized within 12h to 24h, and at 12h, the intracellular polysaccharide and the extracellular polysaccharide respectively act together with LPS, so that the generation of NO in the cells can be remarkably inhibited (P < 0.01). The time for stimulating the cells with LPS in the subsequent experiments was thus set to 12 h.
The effect of cordyceps militaris polysaccharide pre-action on LPS-induced NO secretion by RAW264.7 cells is shown in FIG. 7. As can be seen from FIG. 7, within the concentration range of 0-160 μ g/ml, the intracellular polysaccharide of Cordyceps militaris can significantly inhibit LPS-induced RAW264.7 cells from secreting NO (P <0.01), and the inhibition rates are respectively: 61.42%, 57.64%, 56.48% and 30.76%, and NO content was significantly higher than that of the blank control (P <0.01) only at an intracellular polysaccharide concentration of 160 μ g/ml. Within the concentration range of 0-160 mug/ml, the cordyceps militaris extracellular polysaccharide can remarkably inhibit LPS (LPS-induced RAW 264.7) cells from secreting NO (P is less than 0.01), and the inhibition rates are respectively as follows: 37.96%, 23.56%, 58.80% and 61.11%. When the polysaccharide concentration is 20 and 40 mu g/ml, the NO content is extremely higher than that of a blank control group (P <0.01), and when the polysaccharide concentration is 80-160 mu g/ml, the NO content is not significantly different from that of the blank control group (P > 0.05). The experimental results show that: within the concentration range of 0-160 mu g/ml, the intracellular polysaccharide and the extracellular polysaccharide of the cordyceps militaris respectively pre-act on RAW264.7 cells for 24 hours and can effectively inhibit NO secretion induced by LPS, wherein the inhibition rate of the intracellular polysaccharide (IPCM-2) on NO secretion is totally higher than that of the extracellular polysaccharide (EPCM-2).
The effect of the pre-acting time of the cordyceps militaris polysaccharide on the NO secretion of RAW264.7 cells induced by LPS is shown in FIG. 8. As can be seen from FIG. 8, the NO secretion of RAW264.7 cells pre-acted by 40 μ g/ml of intracellular polysaccharide and extracellular polysaccharide of Cordyceps militaris for 6h is significantly lower than that of LPS-only treated group (P <0.01), and the inhibition rate is 68.32%. When the pre-action time is 12 hours, the cordyceps militaris polysaccharide has NO inhibition effect on NO secretion, and the NO secretion is even higher than that of a group treated by LPS only. When the cordyceps militaris is pre-acted for 24 hours, the cordyceps militaris intracellular and extracellular polysaccharides have inhibition effects on NO secretion, and the inhibition effects are equivalent. Compared with the pre-acting for 6h, the pre-acting for 12h and 24h has the advantages that the effect of the cordyceps militaris intracellular polysaccharide on inhibiting the NO secretion induced by LPS is weakened, and the NO secretion amount changes irregularly along with the change of time. The experimental results show that: 40 mu g/ml of cordyceps militaris intracellular polysaccharide and extracellular polysaccharide respectively pre-act RAW264.7 cells for 6 hours, so that a good inhibition effect on NO secretion induced by LPS can be achieved, and unstable influence factors in the experimental process can be increased due to overlong acting time, so that 6 hours is preferably selected as the good pre-acting time of cordyceps militaris polysaccharide.
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 (10)
1. A preparation method of cordyceps militaris polysaccharide with a function of inhibiting macrophage from secreting NO is characterized by comprising the following steps:
(1) inoculating Cordyceps militaris strains in a liquid culture medium for fermentation culture, and performing solid-liquid separation to obtain Cordyceps militaris fermentation liquor and Cordyceps militaris mycelia;
(2) drying, crushing and sieving the cordyceps militaris mycelia obtained in the step (1) to obtain mycelium powder; then adding petroleum ether for degreasing treatment, and drying to obtain cordyceps militaris mycelium dry powder;
(3) adding the cordyceps militaris mycelium dry powder obtained in the step (2) into water, and performing hot water extraction at the temperature of 65-95 ℃ to obtain cordyceps militaris extract; then concentrating the cordyceps militaris leaching liquor to obtain cordyceps militaris leaching concentrated solution;
(4) removing protein from the cordyceps militaris leaching concentrated solution obtained in the step (3) and the cordyceps militaris fermentation liquor obtained in the step (1) by combining trypsin with a Sevage method respectively to obtain an intracellular crude polysaccharide extracting solution and an extracellular crude polysaccharide extracting solution after protein removal in sequence;
(5) respectively adding the intracellular crude polysaccharide extracting solution and the extracellular crude polysaccharide extracting solution obtained in the step (4) into an ethanol solution, standing, centrifuging, collecting precipitates, and respectively adding water for re-dissolving to obtain an intracellular crude polysaccharide solution and an extracellular polysaccharide solution; decolorizing, dialyzing, concentrating and drying to obtain IPCM and EPCM respectively;
(6) subjecting the IPCM and the EPCM obtained in the step (5) to DEAE-cellulose column chromatography purification, then dialyzing, and freeze-drying to obtain IPCM-1 and EPCM-1; the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO is obtained; wherein, DEAE-cellulose column chromatography adopts Tris-HCL buffer solution with pH 7.2 and containing 0.4mol/L NaCl to elute IPCM, and adopts Tris-HCL buffer solution with pH8.0 and containing 1mol/L NaCl to elute EPCM.
2. The method for preparing cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO according to claim 1, which is characterized in that:
the DEAE-cellulose column chromatography described in the step (6) is carried out by the following steps:
s1, column packing: adding 1/3 column volume of deionized water into the chromatographic column, opening a liquid outlet at the lower end, and pouring DEAE-cellulose filler into the chromatographic column to naturally settle in the chromatographic column, wherein the volume of the filler is 2/3 of the column volume;
s2, balancing: pumping the Tris-HCl buffer solution into the chromatographic column by using a constant flow pump, and opening a liquid outlet at the lower end until the pH value of an effluent liquid is the same as that of the Tris-HCl buffer solution;
s3, loading and elution:
s31, dissolving IPCM in Tris-HCL with the pH value of 8.0, centrifuging, and taking supernatant to obtain IPCM solution; adding IPCM solution into chromatographic column, eluting with Tris-HCl buffer solution with pH of 7.2 and containing 0.4mol/L NaCl, and collecting intracellular polysaccharide of Cordyceps militaris; measuring total sugar content of intracellular polysaccharide of Cordyceps militaris by phenol-sulfuric acid method, mixing single absorption peak samples, dialyzing, concentrating, and freeze drying to obtain IPCM-1;
s32, dissolving the EPCM in a Tris-HCL buffer solution with the pH value of 8.2, centrifuging, and taking a supernatant to obtain an EPCM solution; then adding the EPCM solution into a chromatographic column, eluting with a Tris-HCl buffer solution with the pH value of 8.0 and containing 1mol/L NaCl, and collecting cordyceps militaris extracellular polysaccharide; measuring total sugar content of intracellular polysaccharide of Cordyceps militaris by phenol-sulfuric acid method, mixing single absorption peak samples, dialyzing, concentrating, and freeze drying to obtain EPCM-1.
3. The method for preparing cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO according to claim 2, which is characterized in that:
the specification of the chromatography column described in step S1 is: 3.0cm × 80 cm;
the pH value of the Tris-HCl buffer solution in the step S2 is 7.0-7.6;
the flow rate of the effluent in the step S2 is 1 mL/min;
the concentration of the IPCM solution in the step S31 is 10-15 mg/mL;
the conditions of centrifugation in step S31 and step S32 are: centrifuging at 5000-8000 rpm for 10 min;
the concentration of the EPCM solution in the step S32 is 10-15 mg/mL;
the flow rate of elution described in step S31 and step S32 was 1 mL/min.
4. The method for preparing cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO according to claim 1, which is characterized in that:
the formula of the liquid fermentation medium in the step (1) is as follows: sucrose 50g, KNO34g、KH2PO41g、Mg SO4·7H2O1 g, vitamin B10.05g, pure water supplement to 1000mL, and pH value adjusted to 6.5;
the solid-liquid separation in the step (1) is separation by adopting a gauze filtering, centrifuging or suction filtration mode;
the drying temperature in the step (2) is 50-70 ℃;
sieving in the step (2) is to pass through a sieve of 40-60 meshes;
the solid-to-liquid ratio of the mycelium powder to the petroleum ether in the step (2) is 1: 1-2 g/mL;
the time of degreasing in the step (2) is 2-4 days;
the degreasing treatment in the step (2) is carried out for 1-3 times;
the feed-liquid ratio of the cordyceps militaris mycelium dry powder to water in the step (3) is 1: 20-40, and g/mL;
the extraction time in the step (3) is 1-3 h;
the extraction times in the step (3) are 1-3 times;
the concentration temperature in the step (3) is 50-70 ℃;
the concentration in the step (3) is to concentrate the cordyceps militaris to 1/5-1/10 of the volume of the cordyceps militaris leaching liquor;
the volume ratio of the ethanol solution to the deproteinized intracellular crude polysaccharide extracting solution in the step (5) is 2-5: 1;
the volume ratio of the ethanol solution in the step (5) to the extracellular crude polysaccharide extracting solution except the protein is 2-5: 1;
the ethanol solution in the step (5) is an ethanol solution with a volume fraction of 95%;
the standing condition in the step (5) is as follows: standing for 24-48 h at 0-4 ℃;
the centrifugation conditions in the step (5) are as follows: centrifuging at 5000-8000 rpm for 10-15 min;
the decolorization in the step (5) is performed by adopting H2O2Decoloring the solution;
the concentration of the intracellular crude polysaccharide solution in the step (5) is 15-30 mg/mL;
the concentration of the extracellular polysaccharide solution in the step (5) is 15-30 mg/mL;
the decoloring conditions in the step (5) are as follows: preserving heat for 1-2 h in a 50 ℃ water bath;
the dialysis in the step (5) is carried out by adopting a dialysis bag with the molecular weight cutoff of 6000-8000 Da;
concentrating the cordyceps militaris polysaccharide extract to 1/3-1/5 of the cordyceps militaris polysaccharide extract in volume in the step (5);
and (4) dialyzing in the step (6) by adopting a dialysis bag with the molecular weight cutoff of 6000-8000 Da.
5. The method for preparing cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO according to claim 1, which is characterized in that: further comprising a step of purifying the IPCM-1 and EPCM-1 obtained by the step (6) with a crosslinked Sephadex G-100 gel column; the method specifically comprises the following steps:
(i) adding 1/3 column volume of deionized water into the chromatographic column, opening a liquid outlet at the lower end, filling the swelled crosslinked Sephadex G-100 gel into the chromatographic column, naturally settling the gel in the chromatographic column, and balancing the gel with distilled water for 2-3 hours after filling;
(ii) respectively dissolving IPCM-1 and EPCM-1 in water to obtain IPCM-1 solution and EPCM-1 solution; then respectively adding the IPCM-1 solution and the EPCM-1 solution into a chromatographic column by using a constant flow pump, eluting by using ultrapure water, controlling the flow rate at 1mL/min, measuring the total sugar content by using a phenol-sulfuric acid method, combining single absorption peaks, concentrating and carrying out vacuum freeze drying treatment to obtain purified cordyceps militaris intracellular polysaccharide and cordyceps militaris extracellular polysaccharide;
the concentration of the IPCM-1 solution in the step (ii) is 10-15 mg/mL;
the concentration of the EPCM-1 solution in the step (ii) is 10-15 mg/mL.
6. The method for preparing cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO according to claim 5, wherein the method comprises the following steps:
the number average molecular weight of the intracellular polysaccharide of the cordyceps militaris in the step (ii) is 1.61 multiplied by 104Da, weight average molecular weight 3.25X 104Da; the number average molecular weight of the extracellular polysaccharide of Cordyceps militaris is 1.34 × 104Da with a weight average molecular weight of 2.00X 104Da。
7. A cordyceps militaris polysaccharide with a function of inhibiting macrophage from secreting NO is characterized in that: prepared by the method of any one of claims 1 to 6.
8. The application of the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO in the preparation of anti-inflammatory drugs or health-care food as claimed in claim 7.
9. The application of the cordyceps militaris polysaccharide with the function of inhibiting macrophage from secreting NO as claimed in claim 7 in preparing a medicament for inhibiting macrophage from secreting NO.
10. The use according to claim 9, characterized in that, the method is realized by any one of the following methods:
(A) simultaneously adding cordyceps militaris polysaccharide and lipopolysaccharide into macrophages for cell culture so as to inhibit the macrophages from secreting NO;
(B) adding cordyceps militaris polysaccharide into macrophages, culturing for 6-24 hours, replacing the cordyceps militaris polysaccharide with a culture medium without polysaccharide, and adding LPS to stimulate cells for 12 hours to inhibit the macrophages from secreting NO;
the effective concentration of the cordyceps militaris polysaccharide in the mode (A) is 20-320 mu g/mL;
the culture time in the mode (A) is 6-24 h;
the lipopolysaccharide of the modes (A) and (B) has a concentration of 1. mu.g/ml;
the effective concentration of the cordyceps militaris polysaccharide in the mode (B) is 20-160 mug/mL.
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| CN114292343B (en) * | 2021-12-31 | 2022-11-04 | 华南师范大学 | Preparation method of perennial cerasus extracellular polysaccharide and intracellular polysaccharide and application of perennial cerasus extracellular polysaccharide and intracellular polysaccharide in regulating intestinal microbial flora and reducing blood sugar |
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Application publication date: 20200519 |