CN113150177B - Cordyceps militaris sporocarp alkali extraction heteropolysaccharide and preparation method and application thereof - Google Patents
Cordyceps militaris sporocarp alkali extraction heteropolysaccharide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cordyceps militaris sporocarp alkali extraction heteropolysaccharide, a preparation method and application thereof, and discloses a cordyceps militaris sporocarp alkali extraction heteropolysaccharide with a novel structure for the first time.
Description
Technical Field
The invention relates to a bioactive substance, a preparation method and application thereof, in particular to an alkali extraction heteropolysaccharide in cordyceps militaris sporocarp, a preparation method thereof and application of lipid-lowering.
Background
At present, cardiovascular and cerebrovascular diseases are diseases with higher incidence and death rate than tumors, and are the first diseases threatening human health. The main pathological process is that on the basis of pathological changes of blood vessel walls, components such as denatured low-density lipoprotein (LDL) and the like are gradually deposited under blood vessel intima to cause ischemic or hemorrhagic diseases. Atherosclerosis is the underlying pathology of cardiovascular disease.
Hyperlipidemia is one of the major factors inducing atherosclerosis. When the cholesterol in the body is too high, particularly the low-density lipoprotein level (LDL-C) and triglyceride are increased, oxidized LDL can be phagocytized by macrophages and is deposited in the artery subendothelial skin to cause subendothelial degeneration, so that blood platelets are gathered on the inner wall of the artery, and if the blood platelets are simultaneously damaged by the artery wall or disturbed in cholesterol transportation, lipid spots are easily formed on the artery intima, and finally, the blood vessel wall is raised and protruded to the lumen to form atheromatous spots.
Lipoprotein lipase (LPL) is a key enzyme of lipid metabolism, which is synthesized by extrahepatic parenchymal cells, secreted into the blood, hydrolyzes Triglycerides (TG) on Chylomicrons (CM) and Very Low Density Lipoproteins (VLDL); LPL deficiency causes increased CM and VLDL levels. After being activated, the liver X receptor alpha (LXR alpha) can mediate the expression of a downstream ATP-binding cassette transporter, thereby promoting the efflux of cholesterol and metabolites thereof.
Cordyceps militaris (cordyces militaris L. Link) also called Cordyceps militaris and Cordyceps militaris, which are easy to artificially culture, have become commercialized products. The cordyceps militaris polysaccharide is one of bioactive substances with high cordyceps militaris content, and has various biological activities, such as: enhancing immunity, resisting tumor, resisting virus, resisting aging, and lowering blood sugar. The polysaccharide is prepared from the cordyceps militaris, and the biological activity of the polysaccharide is determined, so that the clinical transformation application and the improvement of the economic added value of the cordyceps militaris polysaccharide can be promoted, and the polysaccharide has important significance on the comprehensive utilization of the cordyceps militaris.
At present, most of cordyceps militaris polysaccharides reported in literatures adopt a water extraction and alcohol precipitation method, but although the method for obtaining the polysaccharides by the alcohol precipitation method is simple in process flow, the obtained polysaccharides are wide in relative molecular mass range and complex in composition, and the method is not beneficial to the explanation of the structure-effect relationship of the polysaccharides and is also not beneficial to the product quality control and the subsequent clinical transformation application.
In recent years, researchers at home and abroad report the lipid-lowering and anti-atherosclerosis effects of the cordyceps militaris extract by using single or combined medicines, and the researchers at home and abroad also report the extraction literature of the cordyceps militaris polysaccharide by water extraction; however, the literature reports of the alkali-extracted polysaccharide of the cordyceps militaris sporocarp are rare, and the structure and the preparation method of the alkali-extracted polysaccharide with the functions of resisting atherosclerosis and reducing lipid are not reported.
Disclosure of Invention
Aiming at the defects, the invention provides a systematic separation method for extracting heteropolysaccharide from cordyceps militaris sporocarp alkali and application thereof, wherein the heteropolysaccharide can be used for preparing medicines for reducing the levels of plasma total cholesterol, triglyceride and low-density lipoprotein cholesterol, improving the activity of plasma lipoprotein esterase and improving the expression of liver X receptor alpha (LXR alpha).
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for extracting heteropolysaccharide from Cordyceps militaris sporocarp by using alkali comprises the following steps:
1) preparing cordyceps militaris sporocarp alkali extraction crude polysaccharide: collecting Cordyceps militaris fruiting body, drying in shade, pulverizing, and breaking cell wall; adding 95% ethanol into the sporophore of wall-broken Cordyceps militaris, reflux extracting, defatting, and filtering to obtain solid substance; adding distilled water into the solid matter, extracting for three times, and centrifuging to obtain residue; extracting the residue with an alkaline water solution, neutralizing with acid, filtering or centrifuging to obtain a supernatant, concentrating under reduced pressure, adding 95% ethanol, standing at 0-4 deg.C overnight, centrifuging, and collecting precipitate; and (4) carrying out vacuum freeze drying on the collected precipitate to obtain the cordyceps militaris sporocarp alkali-extracted crude polysaccharide.
2) Purifying cordyceps militaris sporocarp alkali-extracted polysaccharide: and (3) eluting the cordyceps militaris sporocarp alkali-extracted crude polysaccharide by using 0-2 mol/L sodium chloride through an anion exchange column and separating and purifying the cordyceps militaris sporocarp alkali-extracted crude polysaccharide through a gel permeation chromatography column to obtain pure cordyceps militaris sporocarp heteropolypolysaccharide, which is named as CM 3-W.
The obtained CM3-W, the molecular weight of which is determined by gel chromatography, is characterized in that the molecular weight of the polysaccharide is 19.1 kDa; CM3-W consists of glucose, galactose and mannose; the proportion of each glycosidic bond was determined from the results of methylation analysis and nuclear magnetic resonance spectroscopy (NMR) data. NMR is shown in FIGS. 1-5, and data is shown in Table 1.
Nuclear magnetic resonance spectrum (NMR) data of the obtained cordyceps militaris sporocarp alkali-extracted heteropolysaccharide CM3-W (see table 1):
the mimic structure of CM3-W is as follows:
wherein: glcpIs glucopyranose, ManpIs pyran-type oxymethylmannose, GalpIs galactopyranose, GalfIs a furan type galactose; wherein n is approximately equal to 9, and the polysaccharide is abbreviated as CM 3-W.
Application of Cordyceps militaris sporocarp alkali extracted heteropolysaccharide in preparing medicines for reducing total cholesterol, triglyceride and low density lipoprotein cholesterol levels in blood plasma, and improving activity of lipoprotein lipase and alpha expression of liver X receptor.
The invention discloses the preparation of cordycep militaris sporocarp alkali extraction heteropolysaccharide with a novel structure and the application of the cordycep militaris sporocarp alkali extraction heteropolysaccharide in the aspects of lipid reduction and atherosclerosis resistance for the first time, and provides a detailed implementation case for the structure-activity relationship research of cordycep militaris alkali extraction polysaccharide. In the embodiment of the invention, the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W can obviously reduce the plasma TC, TG and LDL-C levels of apoE (-/-) mice, LDLR (-/-) mice, guinea pigs and hamsters and can up-regulate LPL activity; CM3-W can also significantly reduce lipid accumulation under liver and aortic arch endothelium of an atherosclerosis model apoE (-/-) mouse and an LDLR (-/-) mouse, and has an anti-atherosclerosis effect. In addition, in the aspect of preparation, the implementation method of the invention adopts ion exchange and molecular exclusion chromatography which are commonly used in industrial production, and lays a foundation for the transformation of the invention.
Drawings
FIG. 1 shows the Nuclear Magnetic Resonance (NMR) spectra of 1H (A) and DEPT (B) of alkali-extracted heteropolysaccharide CM3-W from Cordyceps militaris fruiting body.
FIG. 2 shows 2D-NMR spectrum (A) of alkali-extracted heteropolysaccharide CM3-W from Cordyceps militaris fruiting body1H-1H COSY atlas, (B) HSQC NMR atlas, (C) HMBC NMR atlas, and (D) glucoside attribution and structural formula.
FIG. 3 shows the effect of alkali extraction of heteropolysaccharide CM3-W from Cordyceps militaris fruiting body on the formation of atherosclerotic plaques (A and B), plasma cholesterol (TC) level (C), plasma Triglyceride (TG) level (D), distribution of cholesterol (E) in different lipoprotein particles, and distribution of TG (F) in different lipoprotein particles in apoE (-/-) mice. In comparison to the set of models,*P＜0.05；**p is less than 0.01; compared with the simvastatin group, the simvastatin group has the advantages that,##P＜0.01。
FIG. 4 shows the effect of alkali-extracted heteropolysaccharide CM3-W from Cordyceps militaris fruiting body on the activity of lipoprotein esterase LPL (A) and the effect of LXR alpha protein expression. In comparison to the set of models,*P＜0.05；**P＜0.01。
FIG. 5 shows the effect of Cordyceps militaris fruiting body heteropolysaccharide CM3-W on aortic arch lipid accumulation in atherosclerosis model mice.
Detailed Description
The invention is described in detail below with reference to the following figures and specific embodiments:
example 1: preparation of cordyceps militaris sporocarp alkali-extracted heteropolysaccharide CM3-W and application of heteropolysaccharide CM3-W in resisting atherosclerosis
When the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W is extracted and prepared and the lipid-lowering and anti-atherosclerosis effects of the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W on an apolipoprotein E knockout mouse model are verified, the following steps are adopted:
1) preparing cordyceps militaris sporocarp alkali extraction crude polysaccharide: drying Cordyceps militaris fruiting body in shade, pulverizing with plant pulverizer, and sieving with 100 mesh sieve. Adding 95% ethanol into the sporophore of wall-broken Cordyceps militaris, refluxing for 2 hr at a material-to-liquid ratio of 1:6 (W/V), extracting to remove fat and soluble molecules, and filtering to obtain solid. Adding distilled water into the solid, controlling the material-liquid ratio at 1:8 (W/V), heating in water bath at 95 deg.C, extracting for 3 hr, and filtering to remove supernatant. The residue was extracted 2 times. To the residue was added 8 times by volume of 0.5mol/L sodium hydroxide, the water bath heating temperature was controlled at 95 ℃ and the extraction time was 3 hours, and 0.5mol/L hydrochloric acid was added to neutralize the residue under stirring. The alkaline water extraction was repeated twice. Concentrating the extractive solution to one fourth of the original volume, adding 5 times of 95% ethanol, standing at 4 deg.C overnight at 3000 Xg, centrifuging for 15 min, and collecting precipitate. And (4) carrying out vacuum freeze drying on the collected precipitate to obtain the cordyceps militaris sporocarp alkali extraction crude polysaccharide.
2) Purifying cordyceps militaris sporocarp alkali-extracted polysaccharide: subjecting the crude polysaccharide obtained by alkali extraction of the cordyceps militaris sporocarp to DEAE-52 cellulose anion exchange column, eluting with deionized water, collecting eluent, concentrating, dialyzing, and freeze-drying to obtain a semi-finished product of the polysaccharide obtained by alkali extraction of the cordyceps militaris sporocarp. The semi-finished product of the heteropolysaccharide extracted from the cordyceps militaris sporocarp alkali is subjected to Sephadex G-200 Sephadex column chromatography to obtain a finished product CM3-W of the heteropolysaccharide extracted from the cordyceps militaris sporocarp alkali.
3) The lipid-lowering and anti-atherosclerosis application of the cordyceps militaris sporocarp alkali-extracted heteropolysaccharide CM3-W is as follows: SPF-grade apolipoprotein E knockout (apoE-/-) mice, male, weigh 20 + -2 g. The experimental animals were randomized into 3 groups by administering a general feed for 1 week under experimental conditions: model group 10, simvastatin group 10, CM3-W treatment group 10. apoE (-/-) mice were given high fat diet (formula: cholesterol 0.15%, bile salts 0.5%, lard 10%, egg yolk powder 10%, basal diet 78.25%). The corresponding drugs were administered to each group by gavage, and the same volume of physiological saline was administered to the model group 1 time per day for 2 months. The drugs tested and the doses for each group were as follows:
model group: 0.2 mL/kg physiological saline;
simvastatin group: 10 mg/kg simvastatin;
CM 3-group W: 100 mg/kg of cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM 3-W.
During the experiment, no animals died in each group. Fasting for 12h after the last administration, collecting blood of 0.6-0.8 mL of blood from orbital veins by heparin anticoagulation, anesthetizing and killing by pentobarbital sodium, separating plasma, and storing at-80 ℃ for later use; after the residual blood is washed by cardiac injection, the heart and the liver are rapidly picked up, the aortic arch is separated to the whole aorta at the bifurcation of the common iliac artery, fat and connective tissue outside the blood vessel are removed, and microscopic examination is carried out after staining. The resulting serum samples were tested for Total Cholesterol (TC), Triglycerides (TG), low density lipoprotein levels (LDL-C) and lipoprotein lipolytic enzyme (LPL) activity.
The experimental results are shown in figure 3, which shows the influence of the alkali-extracted heteropolysaccharide CM3-W of the fruiting body of Cordyceps militaris on the formation (A and B) of atherosclerotic plaques in apoE (-/-) mice, the level (C) of plasma cholesterol (TC), the level (D) of plasma Triglyceride (TG), the distribution (E) of cholesterol in different lipoprotein particles and the distribution (F) of TG in different lipoprotein particles. In comparison to the set of models,*P＜0.05；**p is less than 0.01; compared with the simvastatin group, the simvastatin group has the advantages that,##p is less than 0.01. The experimental results show that the CM3-W administration group can inhibit the formation of atherosclerotic plaques and can down-regulate TC, TG and LDL-C, and has statistical significance relative to the model group (P is less than 0.01). The experimental results show that: the effect of remarkably reducing high fat induced apoE-/-mouse plasma lipid can be achieved when the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W is 100 mg/kg. Since the lipid-lowering activity of simvastatin requires the involvement of apoE, simvastatin does not exhibit lipid-lowering and anti-atherosclerotic activity in apoE (-/-) mice. Results of simvastatin experiments and experiments already performedThe results of the literature in the table are similar.
FIG. 4 shows the effect of alkali-extracted heteropolysaccharide CM3-W of Cordyceps militaris fruiting body on the activity of lipoprotein esterase LPL (A) and the effect of LXR alpha protein expression. In comparison to the set of models,*P＜0.05；**p is less than 0.01. Experimental results show that CM3-W can significantly up-regulate LPL activity and promote LXR alpha protein expression.
Example 2: preparation of cordyceps militaris sporocarp alkali-extracted heteropolysaccharide CM3-W and application thereof in resisting atherosclerosis
When the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W is extracted and prepared, and the lipid-lowering activity and the anti-atherosclerosis activity of the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W on a low-density lipoprotein receptor knockout (LDLR-/-) mouse model are verified, the following steps are adopted:
1) preparing cordyceps militaris sporocarp alkali extraction crude polysaccharide: drying Cordyceps militaris fruiting body in the shade, pulverizing with plant pulverizer, and sieving with 200 mesh sieve. Adding 95% ethanol into the sporophore of wall-broken Cordyceps militaris, refluxing for 2.5 hr at a material-to-liquid ratio of 1:8 (W/V), extracting to remove fat and soluble molecules, and filtering to obtain solid. Adding distilled water into the solid, performing ultrasonic heating at 400W with a material-liquid ratio of 1:10 (W/V) at 90 deg.C for 4 hr, and filtering to obtain residue. The extraction was repeated 1 time. Adding 9 times of 0.4 mol/L potassium hydroxide into the residue, ultrasonically extracting at 90 deg.C for 4 hr, and adding 0.3 mol/L sulfuric acid for neutralization. The operation was repeated 1 time. Concentrating the extractive solution to one fifth of the original volume, adding 4 times of 95% ethanol, standing at 4 deg.C overnight at 3500 Xg, centrifuging for 20 min, and collecting precipitate. And (4) carrying out vacuum freeze drying on the collected precipitate to obtain the cordyceps militaris sporocarp alkali extraction crude polysaccharide.
2) Purifying cordyceps militaris sporocarp alkali-extracted polysaccharide: the alkali-extracted polysaccharide of the cordyceps militaris sporocarp is firstly eluted by deionized water through a Hiscreen Q FF anion exchange column, and an eluent is collected, concentrated, dialyzed and freeze-dried to obtain a semi-finished product of the alkali-extracted heteropolypolysaccharide of the cordyceps militaris sporocarp. The semi-finished product of heteropolysaccharide extracted from the cordyceps militaris sporocarp by alkali is subjected to Sepharose 6B gel column chromatography to obtain a finished product CM3-W of the heteropolysaccharide of the cordyceps militaris sporocarp by separation.
3) The lipid-lowering and anti-atherosclerosis application of the cordyceps militaris sporocarp alkali-extracted heteropolysaccharide CM3-W is as follows: SPF-grade low density lipoprotein receptor-loaded knockout (LDLR-/-) mice, male, 21 + -2 g in body weight. The experimental animals were randomized into 4 groups by administering a general feed for 1 week under experimental conditions: model group 12, CM3-W low dose group 12, CM3-W medium dose group 12, and CM3-W high dose group 12. Except for the blank group, high fat diet (formula: cholesterol 0.15%, bile salt 0.5%, lard 10%, egg yolk powder 10%, basal diet 78.25%) was administered. The corresponding drug was gavaged 1 time a day for 10 weeks. The drugs tested and the doses for each group were as follows:
model group: 0.2 mL/kg physiological saline;
CM3-W low dose group: 25 mg/kg of cordyceps militaris sporocarp alkali extraction heteropolysaccharide;
CM3-W middle dose group: extracting heteropolysaccharide from 50 mg/kg Cordyceps militaris fruiting body with alkali;
CM3-W high dose group: 100 mg/kg of cordyceps militaris sporocarp alkali extraction heteropolysaccharide.
During the experiment, no animals died in each group. Fasting for 12h after the last administration, collecting blood of 0.6-0.8 mL of blood from orbital veins by heparin anticoagulation, anesthetizing and killing by pentobarbital sodium, separating plasma, and storing at-80 ℃ for later use; after the residual blood is washed by cardiac injection, the heart and the liver are rapidly picked up, the aortic arch is separated to the whole aorta at the bifurcation of the common iliac artery, fat and connective tissue outside the blood vessel are removed, and microscopic examination is carried out after staining. The resulting serum samples were tested for Total Cholesterol (TC), Triglycerides (TG), low density lipoprotein levels (LDL-C) and lipoprotein lipolytic enzyme (LPL) activity.
The experimental results are shown in the table 2, and the influence of the alkali-extracted heteropolysaccharide CM3-W of the cordyceps militaris sporocarp on TC, TG, LDL-C and LPL of LDLR (-/-) mouse plasma is shown, and the experimental results show that TC, TG and LDL-C can be reduced to a certain degree by an administration group, and the activity of LPL is improved. The medium and high dose group of CM3-W had significant effect and had statistical significance (P < 0.01) compared to the model group. The experimental results show that: the effect of remarkably reducing high fat induced LDLR (-/-) mouse plasma lipid can be achieved when the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W is 50 mg/kg.
TABLE 2 influence of Cordyceps militaris fruiting body heteropolysaccharide CM3-W on plasma TC, TG, LDL-C and LPL of LDLR (-/-) mice
| Group of | TC (mg/dl) | TG (mg/dl) | LDL-C (mg/dl) | LPL (U/L) |
| Model set | 886.5 ± 49.5 | 280.42 ± 56.3 | 522.5 ± 63.2 | 36.9 ± 6.1 |
| Low dose | 797.6 ± 53.2* | 260.8 ± 40.7 | 474.3 ± 47.3 | 41.6 ± 5.4 |
| Middle dose | 681.3 ± 41.0** | 227. 4 ± 49.3** | 437.6± 37.9** | 43.1 ± 7.9* |
| High dose | 597.2 ± 81.1 ** | 171.0 ±51.4** | 368.4 ± 52.7** | 54.5 ± 8.9 ** |
P < 0.05 compared to model group; p < 0.01.
As shown in figure 5, the influence of Cordyceps militaris fruiting body heteropolysaccharide CM3-W on aortic arch lipid accumulation of atherosclerosis model mice. The experimental results show that: the administration group, especially the middle and high dose group, can significantly reduce the lipid accumulation under the endothelium of the aortic arch.
Example 3: preparation of cordyceps militaris sporocarp alkali-extracted heteropolysaccharide CM3-W and lipid-lowering application thereof
When the cordyceps militaris sporocarp alkali is extracted and prepared to extract heteropolysaccharide CM3-W and the lipid-lowering activity of the heteropolysaccharide CM3-W on a high-fat induced hamster model is verified, the following steps are adopted:
1) preparing cordyceps militaris sporocarp alkali extraction crude polysaccharide: drying Cordyceps militaris fruiting body in oven at 50 deg.C overnight, pulverizing with plant pulverizer, and sieving with 100 mesh sieve. Adding 95% ethanol into the sporophore of wall-broken Cordyceps militaris, refluxing for 3 hr at a material-to-liquid ratio of 1:6 (W/V), extracting to remove fat and soluble molecules, and filtering to obtain solid. Adding distilled water into the solid at a ratio of 1:12 (W/V), heating in water bath at 90 deg.C for 2.5 hr, and filtering to remove residue. The extraction was repeated 2 times. Adding 0.6 mol/L sodium hydroxide into the residue, heating in 90 deg.C water bath for 4 hr, adding 0.3 mol/L hydrochloric acid, neutralizing under stirring, and repeatedly extracting for 3 times. Combining the alkali extract, concentrating to one third of the original volume, adding 95% ethanol with 6 times of volume into the concentrated solution, standing at 4 deg.C overnight, 4000 Xg, centrifuging for 10 min, and collecting precipitate. And (4) carrying out vacuum freeze drying on the collected precipitate to obtain the cordyceps militaris sporocarp alkali extraction crude polysaccharide.
2) Purifying cordyceps militaris sporocarp polysaccharide: and (3) subjecting the cordyceps militaris sporocarp alkali-extracted polysaccharide to Q-Sepharose Fast Flow anion exchange column, eluting with deionized water, collecting eluent, concentrating, dialyzing, and freeze-drying to obtain a cordyceps militaris sporocarp alkali-extracted heteropolypolysaccharide semi-finished product. The semi-finished product of the heteropolysaccharide extracted from the cordyceps militaris sporocarp alkali is subjected to Sephacryl-S200HR gel column chromatography to obtain a finished product CM3-W of the heteropolysaccharide extracted from the cordyceps militaris sporocarp alkali.
3) The lipid-lowering application of the cordyceps militaris sporocarp heteropolysaccharide CM3-W is as follows: SPF hamster, male, body weight 150. + -.15 g. The experimental animals were randomized into 5 groups by administering a general feed for 1 week under experimental conditions: 9 blank groups, 9 model groups, 9 CM3-W low dose groups, 9 CM3-W medium dose groups, and 9 CM3-W high dose groups. Except for the blank group, high fat diet (formula: cholesterol 1.0%, bile salt 0.5%, lard 10%, egg yolk powder 10%, basal diet 78.25%) was administered; the blank group was given normal feed. The corresponding drugs were administered by gavage, and the same volume of physiological saline was administered 1 time per day for 2 months in the blank group and the model group. The drugs tested and the doses for each group were as follows:
blank group: 0.2 mL/kg physiological saline;
model group: 0.2 mL/kg physiological saline;
CM3-W low dose group: 20 mg/kg of cordyceps militaris sporocarp alkali extraction heteropolysaccharide;
CM3-W middle dose group: extracting heteropolysaccharide from 40 mg/kg Cordyceps militaris fruiting body with alkali;
CM3-W high dose group: 80 mg/kg of cordyceps militaris sporocarp alkali extraction heteropolysaccharide.
During the experiment, no animals died in each group. Fasting is carried out for 12h after the last administration, blood is collected from the orbital vein of the blood vessel by heparin anticoagulation for 5-6 mL, pentobarbital sodium is anesthetized and killed, and plasma is separated and stored at minus 80 ℃ for later use. The resulting serum samples were tested for Total Cholesterol (TC), Triglycerides (TG), low density lipoprotein levels (LDL-C) and lipoprotein esterase (LPL) activity.
Table 3 shows the effect of the alkali extraction of heteropolysaccharide CM3-W from Cordyceps militaris fruiting body on TC, TG, LDL-C and LPL in hamster plasma. The experimental result shows that the TC, TG and LDL-C of the model group are obviously increased relative to the blank control group; the administration group can down-regulate TC, TG and LDL-C and up-regulate LPL. The medium and high dose groups of CM3-W were significantly effective and statistically significant (P < 0.05 or P < 0.01) relative to the model group. The experimental results show that: the effect of remarkably reducing high-fat induced hamster plasma lipid can be achieved when the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W is 40 mg/kg.
TABLE 3 influence of the alkaline extraction of heteropolysaccharide CM3-W from Cordyceps militaris fruiting body on TC, TG, LDL-C and LPL in hamster plasma
| Group of | TC (mg/dl) | TG (mg/dl) | LDL-C (mg/dl) | LPL (U/L) |
| Blank group | 125.3 ± 30.5 | 100.7 ± 20.2 | 83.2 ± 20. 4 | 78.4 ±10.5 |
| Model set | 557.6 ± 89.2 ## | 318.6 ± 70.8 ## | 160.3 ± 71.3 ## | 56.3 ± 5.4 # |
| Low dose | 490.3 ± 84.2 | 294.4 ± 69.3 | 152.9 ± 43.5 | 63.9 ± 10.5 |
| Middle dose | 478.5 ± 69.1* | 270.1 ± 42.8** | 131.3 ± 42.7* | 82.2 ± 16.0** |
| High dose | 353.9 ± 50.4** | 252.5 ± 34.6** | 123.4 ± 39.4** | 84.5 ± 12.3** |
In comparison to the blank set, the data is,#P＜0.05；##p is less than 0.01; p < 0.05 compared to model group; p < 0.01.
Example 4: preparation of cordyceps militaris sporocarp alkali-extracted heteropolysaccharide CM3-W and lipid-lowering application thereof
When the cordyceps militaris sporocarp alkali is extracted and prepared to extract heteropolysaccharide CM3-W and the lipid-lowering activity of the heteropolysaccharide CM3-W on a high-fat induced guinea pig model is verified, the following steps are adopted:
1) preparing cordyceps militaris sporocarp alkali extraction crude polysaccharide: drying the cordyceps militaris sporocarp at 60 ℃, crushing the cordyceps militaris sporocarp by using a plant crusher, and sieving the cordyceps militaris sporocarp by using a 100-mesh sieve. Adding 95% ethanol into the sporophore of wall-broken Cordyceps militaris, refluxing for 4 hr at a material-to-liquid ratio of 1:5 (W/V), extracting to remove fat and soluble molecules, and filtering to obtain solid. Adding distilled water into the solid, performing ultrasonic heating at 500W with a material-liquid ratio of 1:15 (W/V) at 95 deg.C for 2 hr, and filtering to remove residue. The extraction was repeated 3 times. Adding 0.3 mol/L lithium hydroxide into the residue, ultrasonically heating at 500W, controlling the temperature at 95 ℃, extracting for 2 hours, and neutralizing by using 0.2 mol/L nitric acid. Extracting with alkaline water for 2 times, mixing the supernatants, and concentrating under reduced pressure to one fourth of the original volume. To the concentrate was added 4 times the volume of 95% ethanol, left at 6 ℃ overnight at 5000 Xg, centrifuged for 5 minutes, and the precipitate was collected. And (4) carrying out vacuum freeze drying on the collected precipitate to obtain the cordyceps militaris sporocarp alkali extraction crude polysaccharide.
2) Purifying cordyceps militaris sporocarp alkali-extracted polysaccharide: and (3) subjecting the cordyceps militaris sporocarp alkali-extracted polysaccharide to a Sepharose 6 FF anion exchange column, eluting by using deionized water, collecting eluent, and concentrating, dialyzing and freeze-drying to obtain a cordyceps militaris sporocarp alkali-extracted heteropolypolysaccharide semi-finished product. The semi-finished product of the heteropolysaccharide extracted from the cordyceps militaris sporocarp alkali is subjected to QAE Sephadex A-50 gel column chromatography, and a finished product CM3-W of the heteropolysaccharide extracted from the cordyceps militaris sporocarp alkali can be obtained by separation.
3) The lipid-lowering application of the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W is as follows: SPF grade guinea pigs, males, weighing 200 + -15 g. The experimental animals were randomized into 4 groups by administering a general feed for 1 week under experimental conditions: blank 6, model 6, CM3-W low dose 6 and CM3-W high dose 6. Except for the blank group, high fat diet (formula: cholesterol 1.0%, bile salt 0.5%, lard 10%, egg yolk powder 10%, basal diet 77%); the blank group was given normal feed. The corresponding drugs were gavaged, and the same volume of physiological saline was administered to the blank group and the model group 1 time a day for 9 weeks. The drugs tested and the doses for each group were as follows:
blank group: 0.2 mL/kg physiological saline;
model group: 0.2 mL/kg physiological saline;
CM3-W low dose group: 25 mg/kg of cordyceps militaris sporocarp alkali extraction heteropolysaccharide;
CM3-W high dose group: 50 mg/kg of cordyceps militaris sporocarp alkali extraction heteropolysaccharide.
During the experiment, no animals died in each group. Fasting is carried out for 12h after the last administration, blood is collected from the orbital vein of the blood vessel by heparin anticoagulation for 6-8 mL, pentobarbital sodium is anesthetized and killed, and plasma is separated and stored at minus 80 ℃ for later use. The resulting serum samples were tested for Total Cholesterol (TC), Triglycerides (TG), low density lipoprotein levels (LDL-C) and lipoprotein esterase (LPL) activity.
Table 4 shows the effect of alkali extraction of polysaccharide CM3-W from Cordyceps militaris fruiting body on TC, TG, LDL-C and LPL in guinea pig plasma. The experimental results show that the model group has significant increase of TC, TG and LDL-C and significant decrease of LPL relative to the blank control group; the administration group can down-regulate TC, TG and LDL-C and up-regulate LPL activity. The low and high dose groups of CM3-W were significantly effective and statistically significant (P < 0.05 or P < 0.01) relative to the model group. The experimental results show that: the effect of remarkably reducing high-fat induced guinea pig plasma lipid can be achieved when the cordyceps militaris sporocarp alkali extraction heteropolysaccharide CM3-W is 25 mg/kg.
TABLE 4 influence of the alkali extraction of heteropolysaccharide CM3-W from Cordyceps militaris fruiting body on TC, TG, LDL-C and LPL in guinea pig plasma
| Group of | TC (mg/dl) | TG (mg/dl) | LDL-C (mg/dl) | LPL (U/L) |
| Blank group | 132.12± 25.0 | 74.5 ± 8.4 | 78.4 ± 10.3 | 64.2 ±5.8 |
| Model set | 251.1 ± 45.2 ## | 111.3 ±23.7 ## | 151.0 ± 21.5 ## | 48.5 ± 6.3 # |
| Low dose | 210.0 ± 38.3* | 98.8 ±7.4* | 119.2 ± 16.4* | 56.9 ±7.7 |
| High dose | 170.3 ± 26.9** | 93.4 ±8.3** | 98.2± 12.3** | 68.2 ± 10.5* |
Compared with the blank group, # P < 0.05; # P is less than 0.01; p < 0.05 compared to model group; p < 0.01.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art may make modifications, alterations, additions or substitutions within the spirit and scope of the present invention.
Claims (6)
1. A cordyceps militaris sporocarp alkali extraction heteropolysaccharide is characterized in that the structural formula is as follows:
wherein: glcp is glucopyranose, Manp is glucopyranosoxymethyl, Galp is galactopyranose, Galf is galactofuranose; wherein n =9, the polysaccharide is abbreviated as CM 3-W.
2. The cordyceps militaris sporocarp alkali extraction heteropolysaccharide according to claim 1, wherein the molecular weight of the polysaccharide is 19.1 kDa.
3. The method for extracting heteropolysaccharide from cordyceps militaris sporocarp alkali according to claim 1, comprising the following steps:
1) preparing cordyceps militaris sporocarp alkali extraction crude polysaccharide: collecting Cordyceps militaris fruiting body, drying in shade, pulverizing, and breaking cell wall; adding 95% ethanol into the sporophore of wall-broken Cordyceps militaris, reflux extracting, defatting, and filtering to obtain solid substance; adding distilled water into the solid matter, extracting for three times, and centrifuging to obtain residue; extracting the residue with alkaline water solution, neutralizing with acid, filtering or centrifuging to obtain supernatant, concentrating under reduced pressure, adding 95% ethanol, standing at 0-6 deg.C overnight, centrifuging, and collecting precipitate; vacuum freeze drying the collected precipitate to obtain Cordyceps militaris sporocarp alkali-extracted crude polysaccharide;
2) and (3) purifying the cordyceps militaris sporocarp heteropolysaccharide by alkali extraction: the crude polysaccharide extracted from the cordyceps militaris sporocarp by alkali is eluted by 0-2 mol/L sodium chloride through an anion exchange column and is separated and purified by a gel permeation chromatography column, and then pure cordyceps militaris sporocarp alkali-extracted heteropolysaccharide is obtained through separation, and is named as CM 3-W.
4. The use of the cordyceps militaris sporocarp alkali-extracted heteropolysaccharide of claim 1 in the preparation of a medicament for reducing the levels of total cholesterol, triglycerides and low density lipoprotein cholesterol in plasma.
5. The use of the cordyceps militaris sporocarp alkali-extracted heteropolysaccharide of claim 1 in the preparation of a medicament for increasing the activity of superoxide dismutase and lipoprotein lipolytic enzyme, and the level of plasma high density lipoprotein.
6. The use of the cordyceps militaris sporocarp alkali-extracted heteropolysaccharide of claim 1 in the preparation of a medicament for treating atherosclerosis.
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