CN111808210B - Lumbrugua burclover polysaccharide, extraction method and application thereof - Google Patents
Lumbrugua burclover polysaccharide, extraction method and application thereof Download PDFInfo
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- CN111808210B CN111808210B CN202010902235.5A CN202010902235A CN111808210B CN 111808210 B CN111808210 B CN 111808210B CN 202010902235 A CN202010902235 A CN 202010902235A CN 111808210 B CN111808210 B CN 111808210B
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- 230000002588 toxic effect Effects 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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Abstract
The invention provides a lumbricus-fruticosa polysaccharide, an extraction method and application thereof, and relates to the technical field of Tibetan medicine extraction, wherein the polysaccharide comprises the following characteristics: the average molecular weight of the polysaccharide is 3.54 × 104Da and has a repeatable unit structure. The extraction method comprises the following steps: decocting and filtering the wormcast herb to obtain a filtered liquid medicine; stirring and concentrating the filtered liquid medicine to obtain an extract; decocting the extract, and concentrating to obtain semi-finished dry extract; standing the semi-finished dry paste, and drying to obtain the wormcast; sequentially deproteinizing, precipitating with ethanol, refining, purifying, concentrating, and freeze-drying to obtain the above polysaccharide. The application of the wormcast polysaccharide in preparing cosmetics, medicines and foods can provide at least one of the following effects: has the ability of scavenging free radicals; the oxidation resistance is improved; the ability to increase immune activity, including the ability to enhance red blood cell immunoadsorption; the ability to prevent damage caused by ultraviolet radiation and to protect DNA activity.
Description
Technical Field
The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a wormcast nameko polysaccharide, an extraction method and application thereof.
Background
Wormcast (academic name:Torularia humilis（C.A.Mey.）O.E.Schulz) And alias: summer is like that of Ziwu Pu (Jingzhu Ben Cao), Ziwu rubbing Zi (Ganlu Ben Cao Mingming), and is a plant of the family Brassicaceae. The domestic distribution is in various places of Tibet, Qinghai, Gansu, Shanxi, Hebei, inner Mongolia and Xinjiang. Modern medical research shows that the lumbricus-fruticosa has the functions of detoxifying and invigorating stomach, and is mainly used for treating food poisoning, abdominal pain, dyspepsia and the like. The efficacy is described as follows: the four medical classics: "treat food poisoning"; the "Yutuo Bencao": "aiding digestion, detoxication"; the Tibetan medicine prescription of the New edition: treat food poisoning and benefit lung disease.
The plant polysaccharide is a compound formed by connecting a plurality of same or different monosaccharide groups by glycosidic bonds, is commonly present in natural plants, is one of four basic substances forming plant life activities, and is closely related to the maintenance of plant life functions. A great deal of researches in recent years show that the plant polysaccharide has various pharmacological actions of improving the body immunologic function, reducing blood sugar, reducing blood fat, resisting virus, resisting radiation, resisting aging and the like, and has small toxic and side effects on the body. Therefore, the search and extraction of new polysaccharides from natural plants is a new direction for the development of modern medicine and food industries; the research and development of polysaccharides and derivatives thereof are also of great practical significance. Therefore, the method has important reference value and significance for the extraction research and further development of the lumbricus fruticosa polysaccharide.
Disclosure of Invention
The invention aims to provide the wormcast seed polysaccharide which has the functions of scavenging free radicals, resisting oxidation and ultraviolet radiation activity, enhancing immunocompetence, preventing damage caused by ultraviolet radiation, protecting DNA activity and enhancing the red blood cell immunoadsorption capacity.
The technical scheme adopted by the invention for realizing the purpose is as follows:
an lumbricus-alopecuroide polysaccharide, the structural formula is as follows:
The wormcast fruit polysaccharide has the capabilities of scavenging free radicals, resisting oxidation and ultraviolet radiation and enhancing immunocompetence, shows higher capability of scavenging free radicals than ascorbic acid, can prevent damage caused by ultraviolet radiation and protect DNA activity, can improve the capability of resisting pathogens of organisms by enhancing the immunoadsorption capability of red blood cells, and reduces the harm of foreign matters to the organisms.
According to the invention, the lumbricus-fruticosa polysaccharide has a repeatable unit structure, wherein the backbone of the repeating unit structure is (1-4) beta-D-glucose, one → 3) alpha-D-xylose is arranged on the carbon atom at the 6 position of one glucose in the backbone, one → 1) alpha-D-galactose residue is arranged on the carbon atom at the 2 position of the other glucose in the backbone, and the carbon atom at the 3 position of the galactose residue is linked with → 1) alpha-D-galactose. The structure of the lumbricus-fruticosa polysaccharide contains a beta-type pyran ring and an alpha-type pyran ring. The above Pheretima aspergillum polysaccharide has relative molecular mass of 2.14 × 105-2.32×105Da。
According to the invention, the physical properties of the lumbricus bungeana polysaccharide are as follows: the polysaccharide product is light yellow crystal, has no odor, is easily soluble in water, and is insoluble in high concentration organic solvent such as ethanol, benzene, acetone, etc. The water solution after complete dissolution is in a semitransparent state; the iodine-potassium iodide reaction is negative, which indicates that no starch exists; positive in the presence of sugar is indicated by a positive reaction with phenol sulfate.
The invention also aims to provide the application of the lumbricus fruticosa polysaccharide, which comprises the application of the lumbricus fruticosa polysaccharide in the preparation of cosmetics or medicines or foods, wherein the application can provide at least one of the following effects:
-having the ability to scavenge free radicals;
-improving the antioxidant capacity.
The invention is further configured that the lumbricus fruticosa polysaccharide has the capability of improving the immunocompetence, including the capability of enhancing the red blood cell immunoadsorption.
The invention is further configured that the lumbricus fruticosa polysaccharide has the ability of preventing damage caused by ultraviolet radiation and protecting DNA activity.
The invention also aims to provide an extraction method of the wormcast seed polysaccharide, and the obtained product has the capabilities of scavenging free radicals, resisting oxidation and ultraviolet radiation and enhancing immunocompetence.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a method for extracting the lumbricus-fruticosa polysaccharide comprises the following steps:
s1, adding water into a wormcast fruit medicinal material, decocting and filtering to obtain a filtered liquid medicine;
s2, stirring and concentrating the filtered liquid medicine to obtain an extract;
s3, decocting and concentrating the extract, and controlling the decocting temperature according to the density change of the extract to obtain a dry extract semi-finished product;
s4, standing the semi-finished dry paste, taking out, and drying to obtain the wormcast mustard dry paste;
s5, preparing the dried earthworm fruit mustard paste into dispersion liquid, and then performing deproteinization treatment, wherein Sevage reagents for deproteinization are n-butyl alcohol and trichloroacetic acid; and the number of the first and second groups,
and S6, sequentially carrying out alcohol precipitation, refining and purification on the extracting solution obtained by the deproteinization treatment, concentrating and freeze-drying to obtain the wormcast nameko polysaccharides.
According to the invention, in S2, the filtered liquid medicine is stirred and concentrated, the density of the filtered liquid medicine is detected, and when the density of the filtered liquid medicine is not lower than 0.95g/ml, the concentration is completed, so that the wormcast tenuifolia extract is obtained.
According to the invention, the initial temperature for decoction and concentration in S3 is 180-200 ℃; the decoction temperature is controlled according to the density change of the extract, and the method comprises the following specific steps:
when the density of the extract is not lower than 1.02g/ml, the decoction temperature is controlled to be reduced by 10-30 ℃;
when the density of the extract is not lower than 1.10g/ml, the decoction temperature is controlled to be reduced by 0-20 ℃;
when the density of the extract is not less than 1.30g/ml, the decoction temperature is controlled to be reduced by 20-40 ℃, and then the decoction and concentration are finished when the extract is continuously stirred until no steam emerges.
According to the invention, the volume ratio of the dispersion liquid, trichloroacetic acid and n-butanol is 15-25:4:1 in the deproteinization treatment.
In a specific implementation scenario, the deproteinization operation is as follows: adding trichloroacetic acid and n-butanol into the supernatant obtained in the polysaccharide extraction step, mixing uniformly, oscillating the mixed system for 40-60min, centrifuging for 5-10min at the rotation speed of 4000-6000r/min, and taking the supernatant for later use. The volume ratio of the supernatant to the trichloroacetic acid to the n-butanol in the mixed system is 15-25:4: 1. Because the polysaccharide exists in the form of pure sugar chains in plants and also exists in the form of glycoprotein or glycopeptide formed by combining sugar chains and peptide chains, the Sevage reagent can separate the sugar chains from the peptide chains in the protein removal process, thereby removing protein and improving the yield of the polysaccharide.
According to the invention, in the alcohol precipitation operation, a dialysis bag with the molecular weight of 40000Da is adopted for dialysis operation, and the dialysis time is 60-72 h.
In a specific implementation scenario, the alcohol precipitation operation is as follows: adding ethanol with volume fraction of 90-95% into the supernatant obtained by deproteinization, wherein the adding amount is based on that the content of the alcohol in the mixed solution is not less than 85%, uniformly stirring the mixed solution and standing overnight, then centrifuging for 5-10min under the condition that the rotating speed is 4000-5000r/min, then washing the obtained precipitate for 1-2 times by using the ethanol with volume fraction of 90-95%, redissolving the precipitate by using deionized water, transferring the redissolved precipitate into a 40000Da dialysis bag for dialysis for 60-72h, concentrating the trapped fluid, and freeze-drying to obtain the wormcast fruit crude polysaccharide. Because the polysaccharide obtained by extraction is water-soluble polysaccharide, the hydrogen bond between the polysaccharide and water can be destroyed by adding ethanol, the solubility of the polysaccharide in water is reduced, and the polysaccharide is separated out in a precipitation form.
In a specific implementation scenario, the refining operation is as follows: dissolving the crude polysaccharide in distilled water to prepare a crude polysaccharide solution of 5.0g/L, centrifuging, separating the supernatant by DEAE-cellulose column Cl-1 type 30X 2.5cm chromatography, eluting with 0.4mol/L NaCl solution in linear gradient at the flow rate of 0.6mL/min, collecting each eluted polysaccharide fragment (5 mL/tube), detecting by an anthrone-sulfuric acid method until sugar is detected, combining elution peaks, concentrating, dialyzing, freeze-drying to obtain 1 secondary component PS-II, and further purifying.
In a specific implementation scenario, the purification operation is as follows: dissolving PS-II in water to prepare a solution of 10mg/mL, then carrying out chromatographic separation by using a Sephacryl S-400 column 100 multiplied by 2.5cm, carrying out linear gradient elution by using a 0.3mol/L NaCl solution at the elution speed of 1mL/min, collecting each eluted polysaccharide fragment (5 mL/tube), and carrying out tracking detection by using an anthrone-sulfuric acid method until no sugar is detected; mixing the fractions, concentrating, and freeze drying to obtain Pheretima aspergillum polysaccharide TPS-1. Taking a glucose standard sample as a standard, taking phenol-sulfuric acid as a color developing agent, and determining by a colorimetric method to obtain: the purity of TPS-1 polysaccharide was 98.6%.
According to the invention, the wormcast dried paste prepared by S4 can be used for preparing cosmetics, medicines and foods, and the wormcast dried paste takes wormcast polysaccharide as an active ingredient. The obtained wormcast fruit mustard extract in the prior art contains rich moisture, sugar, protein and the like, and provides a good growth and propagation environment for microorganisms, so that the prevention of the pollution of the extract is the key for ensuring the sanitary quality of finished products, and the wormcast fruit mustard is prepared into dry paste, so that the technical problems that the wormcast fruit mustard extract is not beneficial to storage and transportation and the control of the dosage in the prior art can be solved, and the active ingredient wormcast fruit mustard polysaccharide can play a more effective role.
In some embodiments, the process for preparing the wormcast tosomifera extract comprises the steps of S1 and S2. Preferably, the specific implementation method of S1 is as follows: cleaning and slicing a wormcast herb, adding water into the wormcast herb according to a material-liquid ratio of 1kg to 14-16L, carrying out first decoction at 180-200 ℃ for 510-530min at a stirring frequency of 20 min/time, and filtering through a 100-mesh sieve to obtain residue 1 and liquid medicine 1;
adding water into the residue 1 according to the ratio of the residue to the medicinal material of the wormcast fruit mustard of 1kg to 10-11L, carrying out second decoction at the decoction temperature of 180-200 ℃ for 30-50min, wherein the stirring frequency is 20 min/time, then regulating the decoction temperature down by 20 ℃, continuing to decoct for 200-220min, wherein the stirring frequency is 20 min/time, and filtering through a 100-mesh sieve after the decoction is finished to obtain residue 2 and liquid medicine 2; discarding the residue 2, and mixing the medicinal liquid 1 and the medicinal liquid 2 to obtain a filtered medicinal liquid.
Preferably, the specific implementation method of S2 is as follows: and stirring and concentrating the filtered liquid medicine, detecting the density of the filtered liquid medicine, and finishing concentration when the density of the filtered liquid medicine is not lower than 0.95g/ml to obtain the wormcast tenuifolia extract. More preferably, the concentration time is 60-80min, the stirring frequency is 20 min/time, and the decocting temperature is 180-200 deg.C.
In some embodiments, the process for preparing the dried extract of lumbricus includes S3 and S4. Preferably, the specific implementation method of S3 is as follows: the initial temperature for decoction and concentration is 180-200 ℃; controlling the decoction temperature according to the density change of the extract, and specifically comprising the following steps:
when the stirring frequency is 10 min/time and the concentration is 65-85min, and the density of the extract is not lower than 1.02g/ml, controlling the decoction temperature to be reduced by 10-30 ℃;
when the stirring frequency is 10 min/time and the concentration is 20-40min, and the density of the extract is not lower than 1.10g/ml, controlling the decoction temperature to be reduced by 0-20 ℃;
when the density of the extract is not lower than 1.30g/ml after concentrating for 15-25min, controlling the decoction temperature to be reduced by 20-40 ℃, and then continuously stirring until no steam emerges from the extract, and finishing the decoction and concentration.
Preferably, in the decocting and concentrating step, the higher the density of the extract is, the faster the stirring frequency is, the stirring frequency can be properly adjusted according to the density, and when the density of the extract is not lower than 1.30g/ml, the stirring is changed into continuous stirring. The dry paste can be directly prepared without adding powdery pharmaceutic adjuvant by decocting, concentrating and drying, can be directly tableted, filled into capsules or granulated, is favorable for accurately controlling the dosage and has wide market prospect.
It should be noted that the density and temperature control of the extract can be adjusted according to specific implementation scenarios, and other ways of adjusting the decocting temperature according to the density change of the extract belong to the protection scope of the present application. The prepared wormcast dry paste can replace wormcast extract to prepare the Tibetan medicine renqingchangjue. The lumbricus-semen mustards dry paste takes lumbricus-semen mustards polysaccharide as an active ingredient, or is mixed with other medicines for pharmacy, and has the following advantages: 1) storage advantages: the dry paste can be stored conveniently to a great extent, the storage time is prolonged, and the utilization rate of the raw medicinal materials is improved; the defects of troublesome storage of extractum, easy breeding of microorganisms, easy deterioration and the like are avoided. 2) Transportation advantages are as follows: the dry paste can be conveniently transported, and the loss in the transportation process is reduced; meanwhile, the transportation distance and the transportation time can be increased on the premise of ensuring the quality of the medicinal materials. 3) The medicine is accurate: the dry paste can be accurately used according to the prescription, and various parameters such as the dosage, the medication concentration and the like can be accurately controlled, so that the uniformity and the stability of the quality of the medicine can be ensured. The advantages are beneficial to ensuring the stability and effectiveness of the Tibetan medicine, improving the product quality, ensuring the uniform and stable medicine quality, and the extraction method is simple and easy to popularize.
Preferably, the step (4) is implemented as follows: standing the semi-finished dry paste for 4-8h, wherein the drying method is electrothermal forced air drying or vacuum drying or natural drying in the shade. More preferably, the drying temperature of the electric heating forced air drying is 60-80 ℃, and the drying time is 4-8 d. Among the three drying modes, the electro-thermal forced air drying is preferably adopted, so that the problem of long consumption time of natural drying can be avoided, the phenomenon of 'bumping' of the extract in vacuum drying can be avoided easily, and the drug loss caused by the fact that the traditional Chinese medicine fluid extract is splashed to the inner wall of a vacuum drying oven is effectively avoided.
The invention adopts the steps of firstly preparing the wormcast into dry paste and then extracting the wormcast polysaccharide from the dry paste, thereby having the following beneficial effects: 1) the extraction method of the polysaccharide can obtain the polysaccharide with the chemical structure in the invention; the product of the wormcast fruit polysaccharide has higher capability of scavenging free radicals than ascorbic acid, can prevent damage caused by ultraviolet radiation and protect DNA activity, can improve the pathogen resistance of an organism by enhancing the immunoadsorption capability of red blood cells, and reduces the harm of foreign matters to the organism; 2) the product of the wormcast fruit polysaccharide has application value and prospect in the aspects of preparing cosmetics, medicines, foods and the like; 4) according to the invention, when the lumbricus-fruticose mustard polysaccharide is extracted, the lumbricus-fruticose mustard is prepared into dry paste and then is subjected to further fine processing, the preparation of the dry paste of the lumbricus-fruticose mustard is to control the decoction temperature according to the density change of the extract so as to obtain a finished dry paste product, the finished dry paste product takes the lumbricus-fruticose polysaccharide as an active ingredient, can replace the lumbricus-fruticose mustard extract for use, is convenient to store and transport, can accurately control the dosage, improves the stability and effectiveness of the medicine.
Drawings
FIG. 1 is a graph showing the elution profile of crude Pheretima aspergillum polysaccharide from example 1 on a DEAE-cellulose column; the abscissa is the tube number, the left ordinate is the absorbance (490 nm), and the right ordinate is the NaCl concentration (unit: mol/L);
FIG. 2 is an elution profile of PS-II purified in example 1 on a Sephacryls-400 column; the abscissa is the tube number, the left ordinate is the absorbance (490 nm), and the right ordinate is the NaCl concentration (unit: mol/L);
FIG. 3 is a HPAEC profile of hydrolyzed standard monosaccharide and lumbricus polysaccharides of Experimental example 1; a-example 1, B-standard; 1-fucose, 2-rhamnose, 3-arabinose, 4-galactose, 5-glucose, 6-mannose, 7-xylose, 8-fructose;
FIG. 4 is an infrared spectrum of the Arabidopsis thaliana polysaccharide of Experimental example 1; the abscissa is the wave number (unit: cm)-1) And the ordinate is the light transmittance (unit: %);
FIG. 5 is a flow chart of the process for preparing the extract according to the embodiment of the present invention;
FIG. 6 is a flow chart of the process for preparing dry paste in the example of the present invention;
FIG. 7 is a graph showing the density of the extract in example 1 as a function of temperature; the abscissa is density (unit: g/ml), and the ordinate is temperature (unit: DEG C);
FIG. 8 shows the reducing power of the lumbricus bulgaricus polysaccharide and ascorbic acid in Experimental example 2; a-positive control group, B-example 1; the abscissa is concentration (unit: mg/ml) and the ordinate is absorbance (700 nm);
FIG. 9 shows the clearance of hydroxyl radicals by the lumbricus fruticosa polysaccharide and ascorbic acid in Experimental example 2; a-positive control group, B-example 1; the abscissa is concentration (unit: mg/ml) and the ordinate is absorbance (700 nm);
FIG. 10 shows the treatment of chicken red blood cells C by different treatment groups in Experimental example 33bInfluence of receptor rosette rate; a-blank control group, B-saline group, C-example 1; the abscissa is the age of the day(unit: day) with chicken red blood cell C as ordinate3bReceptor rosette rate (unit:%);
FIG. 11 is a graph showing the effect of different treatment groups on the IC rosette rate of chicken erythrocytes in Experimental example 3; a-blank control group, B-saline group, C-example 1; the abscissa is the age in days (unit: day) and the ordinate is the IC rosette rate of chicken red blood cells (unit:%);
FIG. 12 is a graph showing the effect of the lumbricus fruticosa polysaccharide prepared in example 1 on the DNA strand cleavage of the pBR322 plasmid induced by ultraviolet radiation.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
example 1:
a method for extracting Pheretima aspergillum polysaccharide, as shown in FIGS. 5 and 6, comprises the following steps:
(1) cleaning and slicing 4.4kg of wormcast, adding 66L of water into a jacketed kettle with the capacity of 500L, carrying out first decoction at the decoction temperature of 190 ℃ for 520min, stirring at the frequency of 20 min/time, carrying out first filtration after decoction, and sieving with a 100-mesh sieve to obtain medicine residue 1 and medicine liquid 1;
(2) adding 44L of water into the residue 1, decocting for the second time at 180 deg.C for 50min under stirring at 20 min/time, lowering the decocting temperature by 20 deg.C, decocting at 160 deg.C for 220min under stirring at 20 min/time, and filtering with 100 mesh sieve to obtain residue 2 and medicinal liquid 2; discarding the residue 2, and mixing the medicinal liquid 1 and the medicinal liquid 2 to obtain a filtered medicinal liquid;
(3) adding the filtered liquid medicine into a 300L jacketed kettle, stirring and concentrating, setting the concentration temperature to be 180 ℃, stirring for 20 min/time, detecting the density of the filtered liquid medicine, when the density of the filtered liquid medicine is not lower than 0.95g/ml, the concentration time reaches 70min, and completing the concentration to obtain the wormcast fruit mustard extract;
(4) adding the extract into a 300L jacketed kettle, decocting and concentrating, setting initial temperature at 180 deg.C, starting decocting and concentrating, controlling decocting temperature according to density variation of the extract, and making the density variation curve of the extract along with temperature variation as shown in FIG. 7, specifically comprising the following steps:
when the stirring frequency is 10 min/time and the concentration is 75min, the density of the extract reaches 1.02g/ml, and the decoction temperature is adjusted and controlled to be reduced to 150 ℃;
when the stirring frequency is 10 min/time and the concentration is 40min, the density of the extract reaches 1.10g/ml, the decoction temperature is adjusted and controlled to be reduced to 140 ℃,
continuously stirring and concentrating for 15min until the density of the extract reaches 1.30g/ml, and regulating and controlling the decoction temperature to be 100 ℃; then continuously stirring until no steam emerges from the extract, and finishing decoction and concentration to obtain a dry paste semi-finished product;
(5) standing the semi-finished product of the dry paste for 6h, taking out, drying by electric heat forced air at 70 deg.C for 7 days, and collecting paste to obtain dried lumbruellia bracteata extract;
(6) deproteinization: adding 5 times of distilled water into the wormcast paste, stirring to form uniform dispersion liquid, then adding trichloroacetic acid and n-butyl alcohol into the dispersion liquid, uniformly mixing, then oscillating the mixed system for 60min, centrifuging for 10min under the condition that the rotating speed is 5000r/min, and taking supernatant for later use; the volume ratio of the dispersion liquid, the trichloroacetic acid and the n-butyl alcohol in the mixed system is 25:4: 1;
(7) alcohol precipitation: adding 95% ethanol by volume fraction into the supernatant obtained by deproteinization, wherein the addition amount is based on that the content of the alcohol in the mixed solution is not lower than 85%, uniformly stirring the mixed solution and standing overnight, then centrifuging for 5min under the condition that the rotating speed is 5000r/min, then washing the obtained precipitate for 2 times by using 90% ethanol by volume fraction, redissolving the precipitate by using deionized water, transferring the redissolved precipitate into a 40000Da dialysis bag for dialysis for 68h, concentrating the trapped fluid, and freeze-drying to obtain the wormcast fruit mustard crude polysaccharide;
(8) refining: dissolving crude polysaccharide in distilled water to prepare 5.0g/L crude polysaccharide solution, centrifuging, separating the supernatant with DEAE-cellulose column Cl-1 type 30 × 2.5cm chromatography, sequentially eluting with 0, 0.2, 0.4, 0.8 and 1.2mol/L NaCl solution at flow rate of 0.6mL/min, respectively collecting each eluted polysaccharide fragment (5 mL/tube), tracking and detecting with anthrone-sulfuric acid method until no sugar is detected, detecting with partition tube for absorbance value at 490nm, setting the abscissa as collection tube number and the ordinate as absorbance value according to the detection result, making elution curve (as figure 1), combining the same elution peaks, concentrating, dialyzing and freeze drying to obtain 2 secondary components PS-I and PS-II; enriching main polysaccharide components PS-II, and further purifying;
(9) and (3) purification: dissolving PS-II in water to prepare a solution of 10mg/mL, carrying out chromatographic separation by using a Sephacryl S-400 column with the concentration of 100 multiplied by 2.5cm, sequentially carrying out linear gradient elution by using NaCl solutions of 0, 0.1, 0.25, 0.3 and 0.5mol/L at the elution speed of 1mL/min, respectively collecting each eluted polysaccharide fragment (5 mL/tube), and carrying out tracking detection by an anthrone-sulfuric acid method until no sugar is detected; detecting absorbance value at 490nm with septal duct, making elution curve (as figure 2), mixing same fractions according to detection result, concentrating, and freeze drying to obtain Pheretima aspergillum polysaccharide TPS-1. Taking a glucose standard sample as a standard, taking phenol-sulfuric acid as a color developing agent, and determining by a colorimetric method to obtain: the purity of the TPS-1 polysaccharide was 98.6%.
Example 2:
in order to reduce the loss of polysaccharide in the deproteinization step in the extraction method and better control the polysaccharide loss rate and the protein removal rate, the deproteinization step is optimized, and the specific measures are as follows: adding hydroxyacetone and beta-boswellic acid into Sevage reagent for deproteinization, uniformly mixing, adding into dispersion liquid formed by the wormcast fruit dry paste, uniformly mixing, oscillating the mixed system for 40-60min, centrifuging for 5-10min under the condition of the rotating speed of 4000-6000r/min, and taking supernatant for later use; the addition amounts of the hydroxyacetone and the beta-boswellic acid are respectively 0.01-0.1 percent and 0.01-0.15 percent of the weight of the n-butyl alcohol. The existence of the hydroxyacetone and the beta-boswellic acid can promote the protein to be denatured into jelly in the Sevage reagent, reduce the combination firmness degree of glycopeptide, reduce the separation degree of the protein and polysaccharide, reduce the actual dosage and loss of the reagent and reduce the production cost; but also can reduce the loss rate of polysaccharide to be not more than 15 percent, improve the yield of crude polysaccharide and the removal rate of protein, weaken the damage degree of the deproteinization step to the polysaccharide, ensure that the obtained product has high quality and good quality, and improve the production efficiency and the utilization rate of raw materials.
Specifically, in this embodiment, the extraction method of the wormcast versicolor polysaccharide is basically the same as the extraction step in embodiment 1, and the difference is that: in the deproteinization step (6), adding hydroxyacetone and beta-boswellic acid into a Sevage reagent for deproteinization, uniformly mixing, adding the Sevage reagent into a dispersion liquid formed by the dried extract of wormcast and uniformly mixing, oscillating the mixed system for 60min, centrifuging for 10min under the condition that the rotating speed is 5000r/min, and taking a supernatant for later use; the volume ratio of the dispersion liquid, the trichloroacetic acid and the n-butyl alcohol in the mixed system is 25:4: 1; the addition amounts of the hydroxyacetone and the beta-boswellic acid are respectively 0.07% and 0.14% of the weight of the n-butanol.
Example 3:
the extraction method of the wormcast polycarpa polysaccharide is basically the same as the extraction steps in the example 2, and the difference is that: in the deproteinization in the step (6), hydroxyacetone is added into Sevage reagent used for deproteinization, and beta-boswellic acid is not added
Example 4:
the extraction method of the wormcast polycarpa polysaccharide is basically the same as the extraction steps in the example 2, and the difference is that: in the deproteinization in the step (6), beta-boswellic acid is added into a Sevage reagent used for deproteinization, and hydroxyacetone is not added.
Experimental example 1:
the performance of the lumbricus-fruticosa polysaccharide TPS-1 is determined as follows:
1) determination of molecular weight: a sample of the polysaccharide TPS-1 prepared in example 1 (5mg/mL, 200. mu.L) was filtered through a syringe filter (pore size 0.22 μm); the filtered TPS-1 sample was injected into two Ultrahydrogel TM linear tandem columns (ID 7.8 mm. times.300 mm) using NaNO3Eluting the solution (0.1M) at the flow rate of 0.9mL/min to obtain response value data; the data obtained were collected and processed with Empower (Waters, USA) and the molecular weight (Mw) was calculated directly from the molecular weight per elution volume and the defined Mw of the RI signal value.
The measurement results are as follows: average molecular weights of the wormcast seed polysaccharides prepared in example 1Is 2.25X 105Da; the chromatogram of the lumbricus fruticosa polysaccharide TPS-1 shows a single symmetrical narrow peak, which indicates that the lumbricus fruticosa polysaccharide TPS-1 is a homogeneous material; meanwhile, no peaks were detected from the UV detector at 220, 260, 280nm, indicating that the lumbricus frugiperda polysaccharide TPS-1 is free of protein or nucleic acid.
2) Determination of monosaccharide composition: taking the polysaccharide TPS-1 prepared in example 1, respectively hydrolyzing 5mg of the collected polysaccharide TPS-1 with 10mL of trifluoroacetic acid (TFA) (2M) at 110 ℃ for 6 h; removing TFA from TPS-1 by evaporation under reduced pressure, washing with methanol for 3 times, and vacuum drying at 40 deg.C; the monosaccharide composition and monosaccharide content of the hydrolysate were determined by High Performance Anion Exchange Chromatography (HPAEC) together with a pulsed amperometric detector (Thermo Scientific, USA) and a CarboPac PA20 column (ID3mm × 150mm) using standard fucose, rhamnose, arabinose, galactose, xylose, glucose, mannose and fructose as controls;
wherein the HPAEC operating conditions are as follows: mobile phase A: 50mmol/L NaOH; mobile phase B: 1mol/L of NaAc; flow rate: 0.45 mL/min; the gradient elution conditions were as follows: 0-21.1min, 98.2% of A and 1.8% of B; 21.1-30min, 93.2% of A and 6.8% of B; 30-30.1min, 78.2% of A and 21.8% of B; 30.1-50min, 20% A and 80% B. The results are shown in FIG. 3.
FIG. 3 is a HPAEC profile of a hydrolyzed standard monosaccharide and lumbricus coat polysaccharide, A-example 1, B-standard; 1-fucose, 2-rhamnose, 3-arabinose, 4-galactose, 5-glucose, 6-mannose, 7-xylose and 8-fructose. The measurement result is compared with the standard substance, and the results show that: the lumbricus bulgaricus polysaccharide of example 1 peaked at retention times consistent with standards; quantitative analysis by area normalization method revealed that the wormseed mustard polysaccharide of example 1 consists of glucose, galactose and xylose in a ratio of 3:2: 1.
3) Infrared spectrum: mixing dried TPS-12.0 mg of polysaccharide obtained in example 1 with 200mg of KBr powder, grinding in agate mortar, carefully placing on filter paper, pressing into sheet with tablet press, and measuring with Fourier infrared spectrometer at 400-4000cm-1Infrared spectrum scanning is carried out in the range of (1) and an infrared spectrogram is recorded. As shown in fig. 4.
The results are shownThe wormcast fruit polysaccharide shows 3416.38cm in the infrared spectrum-1The broad absorption peaks of (a) are designated as O-H and N-H stretching vibration peaks, and intra-and intermolecular hydrogen bonds exist. 2932.24cm-1Designated as-CH 2, -CH 3. 2193.75cm-1Designated as-CH 2, -CH 3. 1652.95cm-1Designated C-O, C-C peak of oscillation. 1116.89cm-1、1074.63cm-1At 1200-1000cm-1The range is designated as C-O-H stretching vibration in-COOH and C-O-C stretching vibration of ether bond in pyran ring. 895.05cm-1Indicating that the lumbricus fruticosa polysaccharide has beta-type pyran rings. 703.10cm-1Indicating that the lumbricus fruticosa polysaccharide has alpha-type pyran ring. At 1730cm-1The characteristic absorption peak of uronic acid is absent at the left and right, and is 810cm-1And 870cm-1There was also no characteristic absorption peak for mannose, indicating the absence of uronic acid and mannose.
Experimental example 2:
determination of antioxidant and free radical scavenging capacity of lumbricus-fruticosa polysaccharide
1) Determination of the total reducing power: the polysaccharide of the wormcast prepared in example 1 is taken as an experimental sample. Preparing the samples into solutions with the concentration of 1.0mg/mL by using distilled water respectively, then diluting the solutions into different concentration gradients (0.05, 0.25, 0.5, 0.75 and 1.0 mg/mL), adding 25 muL of phosphate buffer solution with the pH value of 6.6 and 25 muL of 1% potassium ferricyanide into 10 muL of polysaccharide solutions with the concentrations respectively, uniformly mixing the solutions, placing the mixture into a 50 ℃ constant temperature incubator for heat preservation for 20min, cooling the mixture, adding 25 muL of 10% trichloroacetic acid, adding 85 muL of distilled water and 17 muL of ferric trichloride. After the reaction at normal temperature, the light absorption value is measured at 700nm by an enzyme-labeling instrument, and the light absorption value is parallel for 3 times, and the average value is taken. And replacing the lumbricus-fruticosa polysaccharide solution with equal concentration and quantity of ascorbic acid as positive control. The results are shown in FIG. 8.
FIG. 8 shows the reducing power of the lumbricus bulgaricus polysaccharide and ascorbic acid; a-positive control group, B-example 1. The higher the absorbance, the more reductive the substance. The results show that: with the increase of the concentration, the absorbance of the lumbricus bungeana polysaccharide and the ascorbic acid shows an ascending trend, the reducing capability is gradually enhanced, and certain dose dependence is shown; at a concentration of 0.25mg/mL, the reducing power of example 1 was slightly weaker than that of ascorbic acid, and slightly stronger than that of ascorbic acid was exhibited in the latter stage. Also shows that the lumbricus fruticosa polysaccharide has high hydrogen supply capability and can react with free radicals to terminate the chain reaction of the free radicals.
2) Determination of the ability to scavenge hydroxyl radicals: the polysaccharide of the wormcast prepared in example 1 is taken as an experimental sample. The reaction system was mixed by dissolving in water at 37 ℃ for 60 minutes and measuring the absorbance at 510nm, wherein the reaction system consisted of 1mL of 75mM FeSO41mL of 6mM sodium salicylate, 1mL of 18mM H2O2The composition of the lumbricus-fruticosa polysaccharide obtained in the embodiment comprises 0.05mg, 0.25mg, 0.5mg, 0.75mg and 1mg by mass respectively; and replacing the lumbricus-fruticosa polysaccharide solution with equal concentration and quantity of ascorbic acid as positive control. Clearance (%) (1-a/b) × 100; wherein, a is a sample, and b is a control. The results are shown in FIG. 9.
FIG. 9 shows the clearance of hydroxyl radicals by the lumbriculus seed polysaccharide and ascorbic acid, A-positive control group, B-example 1. The results show that: both the lumbricus bulgaricus polysaccharide and ascorbic acid showed dose-dependence; the hydroxyl radical clearance rate of example 1 is increased from 35.6% to 92.6%, and the hydroxyl radical clearance rate of ascorbic acid is increased from 5.9% to 63.8%, which shows that the hydroxyl radical clearance capacity of the wormcast toseila polysaccharide of example 1 is much higher than that of ascorbic acid, and damage caused by hydroxyl radicals can be prevented.
Experimental example 3:
determination of immunocompetence-enhancing capability of lumbricus-fructicum polysaccharide
The experimental method comprises the following steps: the polysaccharide of the wormcast prepared in example 1 is taken as an experimental sample. Healthy Liangfeng green-foot partridge chickens of 1 day old are taken and clinically checked for health. Test chickens were randomly divided into 3 groups at 7 days of age, each group having 60 feathers and half female and half male. A is blank control group, and no immunization is carried out on newcastle disease live vaccine; B. all test chickens in group C were immunized by dropping nose and eye-drop with newcastle disease live vaccine (LaSota strain) at 7 days of age, and immunized by twice with newcastle disease vaccine (CS2 strain) at 27 days of age, wherein they were injected subcutaneously (group B was physiological saline group, group C was 25mg/mL of polysaccharide of example 1), 0.2 mL/feather, and 7 days of continuous injection, respectively at 8 days of age. 5 groups of chickens adopt the same feeding method, feeding conditions, environment and feedAnd (5) performing conventional breeding for quality and breeding management. At 14, 21, 28 and 35 days of age, 2 chickens were randomly drawn for each replicate, 8 chickens per group, blood was collected from the heart, anticoagulated with 1% heparin, and tested. Detecting by the method of literature [ influence of compound Chinese medicinal mixture on immunity of chicken red blood cell (junk, Zhang Gui Zhi, Chinese veterinary journal, 2014,50 (6): 58-60), and measuring red blood cell C3bReceptor rosette rate, erythrocyte IC rosette rate. The results are shown in FIGS. 10 and 11.
FIG. 10 shows the treatment of chicken red blood cells C3bInfluence of receptor rosette rate; FIG. 11 is a graph showing the effect of different treatment groups on chicken red blood cell IC rosette rate; a-blank control group, B-saline group, C-example 1. The results show that the red blood cells C of the group B are obtained after the healthy chickens are immunized by the live vaccine of the Newcastle disease3bThe receptor rosette rate and the erythrocyte IC rosette rate are transiently and obviously reduced compared with the group A, which indicates that the immunization with the Newcastle disease live vaccine can cause the transient reduction of the immune function of the erythrocytes of the body. The C group can improve the red blood cell C of the chicken to different degrees at different ages in days3bThe receptor rosette rate and red blood cell IC rosette rate were significantly or very significantly different from group B. The application shows that the wormcast purpurea polysaccharide can obviously improve the immune function of red blood cells, overcome the adverse reaction of transient decline of the immune function of the red blood cells caused by vaccination, improve the pathogen resistance of organisms by enhancing the immune adsorption capacity of the red blood cells and reduce the harm of foreign matters to the organisms.
Experimental example 4:
determination of anti-ultraviolet radiation activity of lumbricus-fruticosa polysaccharide
The experimental method comprises the following steps: the polysaccharide of the wormcast prepared in example 1 is taken as an experimental sample. Mix 2. mu.L of pBR322 plasmid DNA (50. mu.g/mL) with 14. mu.L of PBS and 8. mu.L of polysaccharide (15.6-500. mu.g/mL); the mixture was vortexed and incubated at room temperature under UV lamp irradiation (8W, 20cm) for 20 minutes, then loaded, electrophoresed UV observed and analyzed. The results are shown in FIG. 12.
FIG. 12 is a graph showing the effect of the lumbricus fruticosa polysaccharide prepared in example 1 on the DNA strand cleavage of the pBR322 plasmid induced by ultraviolet radiation. The results show that without any protection, lane 2 is darkest and the plasmid is severely disrupted; the wormcast seed polysaccharide of example 1 has good DNA protection activity, and the effect is best at 125 mug/mL; the results show that the lumbricus fruticosa polysaccharide prepared in example 1 can prevent damage caused by ultraviolet radiation and has the ability to protect DNA activity.
Experimental example 5:
effect of different extraction methods on polysaccharide loss, protein removal and crude polysaccharide yield
The test method comprises the following steps: the above-mentioned extraction methods of examples 1-4 were respectively used to prepare the above-mentioned lumbricus-frutus-mustard polysaccharide. Respectively measuring the protein content and the polysaccharide content in the supernatant before deproteinization and the supernatant after deproteinization, and calculating the protein removal rate and the polysaccharide loss rate according to the following calculation formula: protein removal rate% = (M1-M2)/M1 × 100%; polysaccharide loss% = (N1-N2)/N1 × 100%; in the formula, M1-protein content in the supernatant before deproteinization, M2-protein content in the supernatant after deproteinization, N1-polysaccharide content in the supernatant before deproteinization, and N2-polysaccharide content in the supernatant after deproteinization; the yield (mg/g) of the crude polysaccharide is not less than the mass of the crude polysaccharide/mass of the raw material of the wormcast. The method for measuring the polysaccharide content is a phenol-sulfuric acid method, and the method for measuring the protein content is a Brodford method. The results are shown in Table 1.
TABLE 1 Effect of different deproteinization methods on polysaccharide loss, protein removal and crude polysaccharide yield
| Example 1 | Example 2 | Example 3 | Example 4 | |
| Polysaccharide loss% | 18.9 | 13.6 | 19.7 | 17.4 |
| Protein removal Rate% | 81.4 | 87.9 | 80.2 | 83.4 |
| The yield of crude polysaccharide is mg/g | 48.66 | 51.84 | 48.18 | 49.56 |
The results show that the polysaccharide loss rate difference of the examples 1 and 3 is not obvious, and the protein removal rate of the example 3 is slightly reduced, so that the yield of the example 3 is reduced to a certain extent compared with the example 1, and the negative effect is achieved; example 4 compared with example 1, the performance is improved, the polysaccharide loss rate and the crude polysaccharide yield are improved, and the protein removal rate is reduced; the embodiment 2 has the remarkable beneficial effects that the polysaccharide loss rate is reduced to below 15%, the protein removal rate is improved to above 85%, and the polysaccharide yield is remarkably improved; the results show that the hydroxyacetone and the beta-boswellic acid added in the example 2 can exert a synergistic effect, the damage degree of the deproteinization step to the product polysaccharide is weakened, the obtained product has high quality and good quality, the production efficiency and the raw material utilization rate are improved, the loss and the waste of the raw material can be reduced, and the raw material utilization rate and the production benefit are improved.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit the same; although the present application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not necessarily depart from the spirit and scope of the corresponding technical solutions in the embodiments of the present application. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (2)
1. The average molecular weight of the lumbricus bulgaricus polysaccharide is 2.25 x 105Da; the wormcast is characterized in that the wormcast polysaccharide consists of glucose, galactose and xylose in a ratio of 3:2: 1;
the extraction method of the wormcast comprises the following steps:
s1, adding water into a wormcast fruit medicinal material, decocting and filtering to obtain a filtered liquid medicine;
s2, stirring and concentrating the filtered liquid medicine to obtain an extract; the density of the extract is not lower than 0.95 g/mL;
s3, decocting and concentrating the extract, and controlling the decocting temperature according to the density change of the extract to obtain a dry extract semi-finished product;
s4, standing the semi-finished dry paste, taking out, and drying to obtain the wormcast;
s5, preparing the wormcast dried paste into dispersion liquid, and performing deproteinization treatment by adopting n-butyl alcohol and trichloroacetic acid; the volume ratio of the dispersion liquid to the trichloroacetic acid to the n-butanol is 25:4: 1;
s6, sequentially carrying out alcohol precipitation, dialysis and freeze drying on the extracting solution obtained by deproteinization treatment to obtain the crude lumbricus-fruticosa polysaccharide;
s7, performing column chromatography separation on the crude polysaccharide by using a DEAE-Cellulose column, sequentially performing linear gradient elution by using NaCl solutions of 0 mol/L, 0.2 mol/L, 0.4mol/L, 0.8 mol/L and 1.2mol/L, and combining the same elution peaks to obtain 2 secondary components of PS-I and PS-II;
s8, dissolving PS-II enriched with main polysaccharide components in water, performing column chromatography by using Sephacryl S-400, performing linear gradient elution by using NaCl solutions of 0 mol/L, 0.1 mol/L, 0.25 mol/L, 0.3mol/L and 0.5mol/L in sequence, combining same fractions, concentrating and freeze-drying to obtain the wormcast;
in step S3, the specific steps of controlling the decocting temperature according to the density change of the extract are as follows:
when the stirring frequency is 10 min/time and the concentration is 75min, the density of the extract reaches 1.02g/ml, and the decoction temperature is adjusted and controlled to be reduced to 150 ℃;
when the stirring frequency is 10 min/time and the concentration is 40min, the density of the extract reaches 1.10g/ml, and the decoction temperature is adjusted and controlled to be reduced to 140 ℃;
continuously stirring and concentrating for 15min until the density of the extract reaches 1.30g/ml, and regulating and controlling the decoction temperature to be 100 ℃; and then continuously stirring until no steam emerges from the extract, and finishing decoction and concentration to obtain a dry paste semi-finished product.
2. The use of the lumbricus fruticosa polysaccharide of claim 1 in preparing cosmetics, medicines or foods, is characterized in that: the use can provide at least one of the following effects:
has the ability of scavenging free radicals;
the oxidation resistance is improved;
has the ability to enhance immune activity, including enhancing red blood cell immunoadsorption ability;
has the ability to prevent damage caused by ultraviolet radiation and protect DNA activity.
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| CN105641016A (en) * | 2016-01-25 | 2016-06-08 | 济南星懿医药技术有限公司 | Pharmaceutical composition for treating coronary heart disease |
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| CN105641016A (en) * | 2016-01-25 | 2016-06-08 | 济南星懿医药技术有限公司 | Pharmaceutical composition for treating coronary heart disease |
Non-Patent Citations (2)
| Title |
|---|
| 十字花科植物花蜜腺的比较形态学研究;邓彦斌等;《植物分类学报》;19950610;第33卷(第3期);第209-220页，图版1-3 * |
| 响应面法优化超声提取宽叶独行菜多糖工艺研究;徐传远等;《粮食与油脂》;20150210;第28卷(第2期);第59-62页 * |
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