CN110183542B - Method for extracting phellinus igniarius polysaccharides from poplar trees - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention provides a method for extracting phellinus igniarius polysaccharides from poplar trees, which comprises the steps of crushing phellinus igniarius sporocarp to 1-5 meshes, soaking the phellinus igniarius sporocarp in normal saline for 8-24 hours, centrifuging and collecting residues; then, boiling phellinus igniarius sporocarp residues in boiling water at the temperature of 110-130 ℃ for 20-40 min, centrifuging and collecting residues; further adding water into the residue, freezing for 24-36h, and thawing; adding water into the unfrozen mixture to obtain a mixed solution, adding a complex enzyme into the mixed solution, carrying out enzymolysis for 2-4 h at 50-60 ℃, and carrying out centrifugal separation to obtain a supernatant; and finally, adsorbing the supernatant by using resin, dialyzing, concentrating and freeze-drying to obtain the phellinus igniarius polysaccharides of the poplar. The extraction method of the phellinus igniarius polysaccharide provided by the invention has obvious activity of inhibiting tumor cell proliferation, has good biocompatibility compared with polysaccharide extracted and purified by a chemical method, and does not bring inflammatory reaction.
Description
Technical Field
The invention relates to the technical field of medicine extraction, and particularly relates to an extraction method of phellinus igniarius polysaccharides of poplar trees.
Background
Phellinus linteus is a precious medicinal fungus. The fungus is usually grown on Morus plants in the central and south areas of China, and the fruit body is famous for being fresh and yellow. Phellinus linteus as a traditional Chinese medicine has been in China for thousands of years, and in the past generation of herbal work, Phellinus linteus is also named as sang minister, Morus alba Linn, Phellinus linteus Linn, and the like. Phellinus linteus generally parasitizes on trunks of broad-leaved trees such as mulberry trees, poplar trees, white birch trees, peach trees, willow trees and the like, and the species and the components of the phellinus linteus are different due to different species and different varieties of the trees, so that a large number of scientifically reported phellinus linteus polysaccharides are different in component, and the root cause of the phellinus linteus is to confuse the source of raw materials. Phellinus linteus (Phellinus vanninii Ljup) is a kind of Phellinus linteus, and is originated from Populus linteus. Also known as "Bei Han sang Huang", produced in Changbai mountain of Jilin. At present, the phellinus linteus which is most accepted by korea is the largest in the market, and has the functions of resisting tumor, enhancing immunity, resisting oxidation, protecting liver and the like, and is the strain with the shape most similar to that of the strain cultivated in korea.
The sporocarp of phellinus igniarius is lignified, and the cell wall contains a large amount of chitin and cellulose macromolecules, so that the phellinus igniarius polysaccharide of the poplar is difficult to extract. In the existing research results, the extraction method mainly comprises water extraction and alcohol precipitation, and the extraction rate is low. In the previous research, the degreased phellinus igniarius sporocarp of the poplar is subjected to gradual wall breaking, and two polysaccharides are extracted by hot water and alkali liquor in sequence, wherein the yield of the polysaccharides extracted by the hot water is low, and the biological activity is poor; the yield of polysaccharide extracted by alkali liquor is higher, but in subsequent researches, the alkali extracted polysaccharide can stimulate macrophages to produce a large amount of inflammatory factors, possibly bring inflammatory reaction and cause certain negative effects on the biomedical use.
Therefore, it is an urgent need to solve the problems in the art to provide a method for extracting phellinus igniarius polysaccharides from poplar, which can improve the yield of phellinus igniarius polysaccharides and obtain phellinus igniarius polysaccharides with good biocompatibility.
Disclosure of Invention
The invention is based on at least one of the technical problems, and provides a method for extracting the phellinus igniarius polysaccharides of the poplar, which comprises the steps of repeatedly leaching and removing impurities by normal saline and hot water, and repeatedly freezing and thawing phellinus igniarius sporophore cells by adopting a low-temperature freezing-microwave quick thawing method, so that the cell wall structure of the phellinus igniarius sporophore cells is fully damaged, and the extraction yield of the phellinus igniarius polysaccharides of the poplar is improved; and secondly, the adsorption action of the macroporous resin is adopted to remove protein and pigment, other chemical micromolecules are not introduced under the condition of ensuring the effect of removing impurities to the maximum extent, and the inflammatory reaction caused by the extracted polysaccharide is greatly reduced.
In view of the above, the invention provides a method for extracting phellinus igniarius polysaccharides from poplar, comprising the following steps:
(1) crushing phellinus igniarius sporocarp to 1-5 meshes, soaking the phellinus igniarius sporocarp in normal saline for 8-24 hours, repeating the soaking for 1-3 times, centrifuging and collecting residues;
(2) boiling phellinus igniarius sporocarp residues in boiling water at the temperature of 110-130 ℃ for 20-40 min, repeating for 1-3 times, centrifuging and collecting residues;
(3) adding water into the residue in the step (2), freezing at-15 deg.C to-10 deg.C for 24-36h, and thawing; repeating the freezing-unfreezing process for 1-3 times;
(4) adding water into the unfrozen mixture obtained in the step (3) to obtain a mixed solution, adding a complex enzyme into the mixed solution, carrying out enzymolysis for 2-4 h at 50-60 ℃, and carrying out centrifugal separation to obtain a supernatant;
(5) and adsorbing the supernatant with resin for 1-3 times to remove impurities, and then dialyzing, concentrating and freeze-drying to obtain the poplar phellinus igniarius polysaccharide.
The extraction method of the phellinus igniarius polysaccharides of the poplar provided by the invention is a physical extraction process, other chemical micromolecules are not introduced in the extraction process, the extraction yield of the phellinus igniarius polysaccharides of the poplar is 6.8%, and the extracted phellinus igniarius polysaccharides of the poplar have good biocompatibility, have obvious activity of inhibiting the proliferation of tumor cells and do not bring inflammatory reaction.
Further, the adding amount of the phellinus linteus fruit body residue and boiling water in the step (2) is 60-80 g/L.
Further, the addition amount of the residue and water in the step (3) is 200 g/L.
Further, the thawing method in the step (3) comprises the following steps: the microwave is used for rapid thawing, and the power is 1000-3000W.
Wherein, the method of low-temperature freezing-microwave quick thawing is used for repeatedly freezing and thawing the phellinus igniarius sporophore cells, so as to fully destroy the cell wall structure and improve the extraction yield of the phellinus igniarius polysaccharides of the poplar.
Further, the addition amount of the unfreezing mixture and the water in the step (4) is 60-80 g/L.
Further, the complex enzyme in the step (4) comprises cellulase and chitinase; wherein the mass ratio of the cellulase to the chitinase is 1: 1-3.
Furthermore, the adding amount of the complex enzyme in the step (4) is 0.2 to 0.5 ten thousandth of the weight of the mixed solution.
Further, the resin in the step (5) is HP20 macroporous resin; wherein the mass ratio of the macroporous resin to the supernatant is 1: 20-50.
The adsorption of HP20 macroporous resin is used for deproteinizing and depigmenting, so that other chemical micromolecules are not introduced under the condition of ensuring the effect of removing impurities to the maximum extent, and the inflammatory reaction caused by the extracted polysaccharide is greatly reduced.
Based on the second aspect of the invention, the invention provides the application of the phellinus linteus polysaccharide of the poplar in preparing the antitumor drugs or the health care products.
Through the technical scheme, the phellinus igniarius polysaccharide obtained by the method has obvious activity of inhibiting tumor cell proliferation, has good biocompatibility compared with polysaccharide extracted and purified by a chemical method, does not bring inflammatory reaction, and can be used in antitumor drugs or health care products. The invention also has the following advantages:
(1) the phellinus igniarius polysaccharides of the poplar are derived from a special drug resource, the phellinus igniarius of the poplar has rich yield and low cost;
(2) the polysaccharide extraction process is environment-friendly and green, is basically a physical extraction process, and has an extraction rate of 6.8%;
(3) the polysaccharide extraction process adopts a freeze thawing method, and utilizes the rapid melting and wall breaking of ice crystals, so that the extraction yield is greatly improved;
(4) the polysaccharide has no adverse side effects, and good biocompatibility.
Drawings
FIG. 1 shows the PV-F spectrum of Phellinus linteus polysaccharide of Poplar in example 2 of the present invention.
FIG. 2 shows nuclear magnetic resonance carbon spectrum of Phellinus linteus polysaccharide PV-F in example 2 of the present invention.
FIG. 3 shows the inhibition rate of Hela cell growth by different concentrations of Phellinus linteus polysaccharide PV-F of Populus linteus in example 2 of the present invention.
FIG. 4 shows the inhibition rate of different concentrations of Phellinus linteus polysaccharide PV-F in example 2 of the present invention on the growth of HepG2 cells.
FIG. 5 shows the effect of different concentrations of Phellinus linteus polysaccharide PV-F in example 2 of the present invention on the release amount of NO as an inflammatory factor of macrophage RAW 264.7.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, the present invention will be described in further detail with reference to specific embodiments. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and therefore the scope of the present invention is not limited by the specific embodiments disclosed below.
Example 1
Wherein the commercial Phellinus linteus fruiting body is purchased in Jilin Changbai mountain area.
A method for extracting phellinus igniarius polysaccharides from poplar trees comprises the following steps:
(1) crushing phellinus igniarius sporocarp to 1-5 meshes, soaking the phellinus igniarius sporocarp in normal saline for 8-24 hours, repeating the soaking for 1-3 times, centrifuging and collecting residues;
(2) boiling the phellinus igniarius sporocarp residues in boiling water at the temperature of 110-130 ℃ for 20-40 min, wherein the adding amount of the phellinus igniarius sporocarp residues and the boiling water is 60-80 g/L; repeating for 1-3 times, centrifuging and collecting residues;
(3) adding water into the residue in the step (2), wherein the adding amount of the residue and the water is 200 g/L; freezing at-15 to-10 ℃ for 24 to 36 hours, and then rapidly thawing by microwave with the power of 1000-3000W; repeating the freezing-unfreezing process for 1-3 times;
(4) adding water into the unfreezing mixture obtained in the step (3) at a ratio of 60-80g/L to obtain a mixed solution, adding a complex enzyme with a weight of 02-0.5 ten thousandth of the weight of the mixed solution into the mixed solution, carrying out enzymolysis at 50-60 ℃ for 2-4 h, and carrying out centrifugal separation to obtain a supernatant;
(5) and adsorbing and removing impurities from the supernatant by using HP20 macroporous resin for 1-3 times, wherein the mass ratio of the macroporous resin to the supernatant is 1: 20-50, and dialyzing, concentrating and freeze-drying by using clear water and secondary distilled water to obtain the poplar phellinus igniarius polysaccharide.
Example 2
Taking 100g of poplar yellow fruit bodies, crushing the poplar yellow fruit bodies to 3 meshes, soaking the poplar yellow fruit bodies in normal saline for 24 hours, centrifuging the poplar yellow fruit bodies, and collecting residues; leaching the residue with 1.5L water at 120 deg.C for half an hour, centrifuging, collecting residue, and repeating twice; dissolving the residue in 0.5L deionized water, mixing, freezing at-12 deg.C for 24 hr, rapidly thawing with microwave at power of 3000W until there is no ice, and repeating for three times; continuously adding 1.5 parts by weight of deionized water, adding 0.5 parts by weight of a mixed solution of a complex enzyme, wherein the complex enzyme is cellulase, namely chitinase, in a ratio of 1:3, performing enzymolysis for 4 hours at 50 ℃, and performing centrifugal separation to obtain a supernatant; removing free protein and pigment from the supernatant with HP20 macroporous resin at a mass ratio of 1:20, and repeating for 3 times; dialyzing with clear water and redistilled water (cutoff Mw 8,000), concentrating, and freeze-drying to obtain Phellinus Baumii polysaccharide (PV-F). The calculated yield was 6.8% by weight and the protein content was about 0.25% by elemental analysis (elemental analysis results are shown in table 1).
TABLE 1 Poplar Phellinus Linteus polysaccharide PV-F elemental analysis results
The protein content was calculated from the nitrogen content 6.25, and it can be seen from table 1 that the protein content was low.
The obtained phellinus linteus polysaccharides of poplar were detected by NMR, FT-IR and GC-MS techniques, and the results are shown in Table 2 and FIGS. 1-2, respectively.
TABLE 2 monosaccharide composition of Poplar Phellinus Linteus polysaccharide PV-F
As shown in table 2, the content of monosaccharide components was obtained by gas chromatography, and it was found that glucose was the main monosaccharide component.
As shown in FIG. 1, the infrared shows a single peak at 890cm-1, illustrating the beta glucan structure. The nuclear magnetic results are shown in FIG. 2, where peaks at 103.7(C1), 73.9(C2), 86.9(C3), 68.9(C4), 76.6-77.3(C5) and 61.4(C6) ppm are typical characteristics of β - (1 → 3) -D-glucan, and the peak at 70.5(C6s) ppm is due to the effect of branching on the backbone, indicating that glucan is branched at C6. In summary, PV-F is primarily β - (1 → 3) -D-glucan, containing pendant glucose groups linked by the moiety (1 → 6).
The obtained phellinus linteus polysaccharides of poplar were detected by light scattering detection, and the relevant molecular parameters are shown in table 3.
TABLE 3 monosaccharide composition of Poplar Phellinus Linteus polysaccharide PV-F
Wherein, as shown in Table 3, the weight average molecular weight of Phellinus linteus polysaccharide is about 55 ten thousand, the mean square radius of rotation is 40.8nm, and the Mw and Mw are measured by light scattering detection<s2>z1/2The scatter plot calculation yields α ═ 0.58, indicating that PV-F is a relatively stretched random coil conformation in aqueous solution. The characteristic viscosity of the aqueous solution is 345 calculated by a viscosity method, and the value of the Huggins constant k' in the aqueous solution is 0.37 and is between 0.3 and 0.5, which indicates that the aqueous solution is well dissolved in water.
Example 3
Taking 100g of poplar yellow fruit bodies, crushing the poplar yellow fruit bodies to 2 meshes, soaking the poplar yellow fruit bodies in normal saline for 24 hours, centrifuging the poplar yellow fruit bodies, and collecting residues; leaching the residue with 1.5L water at 120 deg.C for half an hour, centrifuging, collecting residue, and repeating twice; dissolving the residue in 0.5L deionized water, mixing, freezing at-13 deg.C for 30 hr, rapidly thawing with microwave at power of 3000W, and repeating for three times; continuously adding 1.5 parts by weight of deionized water, adding 0.5 parts by weight of a mixed solution of a complex enzyme, wherein the complex enzyme is cellulase, namely chitinase, in a ratio of 1:3, performing enzymolysis for 4 hours at 50 ℃, and performing centrifugal separation to obtain a supernatant; removing free protein and pigment from the supernatant by using HP20 macroporous resin, wherein the mass ratio of the macroporous resin to the supernatant is 1:20, and removing impurities once; dialyzing with clear water and redistilled water (cutoff Mw is 8,000), concentrating, and freeze-drying to obtain Phellinus Baumii polysaccharide. The yield was 7.1% by weight, and the protein content was about 0.65% by elemental analysis, which was low. The identification shows that the chemical structure of the polysaccharide is the same as the poplar yellow polysaccharide PV-F prepared in example 2, and the protein impurities are slightly higher.
Example 4
Taking 100g of poplar yellow fruit bodies, crushing the poplar yellow fruit bodies to 1-5 meshes, soaking the poplar yellow fruit bodies in physiological saline for 24 hours, centrifuging the poplar yellow fruit bodies, and collecting residues; leaching the residue with 1.5L water at 120 deg.C for half an hour, centrifuging, collecting residue, and repeating twice; dissolving the residue in 0.5L deionized water, mixing, freezing at-15 deg.C for 24 hr, and rapidly thawing with microwave at power of 3000W; continuously adding 1.5 parts by weight of deionized water, adding 0.5 parts by weight of a mixed solution of a complex enzyme, wherein the complex enzyme is cellulase, namely chitinase, in a ratio of 1:3, performing enzymolysis for 4 hours at 50 ℃, and performing centrifugal separation to obtain a supernatant; removing free protein and pigment from the supernatant by using HP20 macroporous resin, wherein the mass ratio of the macroporous resin to the supernatant is 1:20, and removing impurities for three times; dialyzing with clear water and redistilled water (cutoff Mw is 8,000), concentrating, and freeze-drying to obtain Phellinus Baumii polysaccharide. The yield calculated by weighing is 4.9%, and is slightly lower than that of example 1 in which freeze thawing is repeated three times, which indicates that the extraction rate can be greatly improved by the freeze thawing method. The protein content detected by elemental analysis was about 0.28%, which was low, similar to example 2. The identification shows that the chemical structure of the polysaccharide is the same as the poplar yellow polysaccharide PV-F prepared in example 1, and the content of protein impurities is close.
Example 5
Taking 100g of poplar yellow fruit bodies, crushing the poplar yellow fruit bodies to 3 meshes, soaking the poplar yellow fruit bodies in normal saline for 24 hours, centrifuging the poplar yellow fruit bodies, and collecting residues; leaching the residue with 1.5L water at 120 deg.C for half an hour, centrifuging, collecting residue, and repeating twice; dissolving the residue in 0.5L deionized water, mixing, freezing at-12 deg.C for 24 hr, rapidly thawing with microwave of 1000W, and repeating for three times; continuously adding 1.5 parts by weight of deionized water, adding 0.5 parts by weight of a mixed solution of a complex enzyme, wherein the complex enzyme is cellulase, namely chitinase, in a ratio of 1:3, performing enzymolysis for 4 hours at 50 ℃, and performing centrifugal separation to obtain a supernatant; removing free protein and pigment from the supernatant with HP20 macroporous resin at a mass ratio of 1:20, and repeating for 3 times; dialyzing with clear water and redistilled water (cutoff Mw 8,000), concentrating, and freeze-drying to obtain Phellinus Baumii polysaccharide (PV-F). The calculated yield was 3.8% by weight, and the protein content was about 0.26% by elemental analysis, which was similar to that of example 2. The chemical structure is identified to be the same as the poplar yellow polysaccharide PV-F prepared in the example 2, and the content of protein impurities is close. The thawing power adopted by the embodiment is 1000W, which is lower than that of the embodiment 2, and the yield is far lower than that of the embodiment 2, which shows that the yield can be greatly improved by improving the thawing speed.
Example 6
Taking 100g of poplar yellow fruit bodies, crushing the poplar yellow fruit bodies to 3 meshes, soaking the poplar yellow fruit bodies in normal saline for 24 hours, centrifuging the poplar yellow fruit bodies, and collecting residues; leaching the residue with 1.5L water at 120 deg.C for half an hour, centrifuging, collecting residue, and repeating twice; dissolving the residue in 0.5L deionized water, mixing, freezing at-4 deg.C for 8 hr, rapidly thawing with microwave at power of 2000W, and repeating for three times; continuously adding 1.5 parts by weight of deionized water, adding 0.5 parts by weight of a mixed solution of a complex enzyme, wherein the complex enzyme is cellulase, namely chitinase, in a ratio of 1:3, performing enzymolysis for 4 hours at 50 ℃, and performing centrifugal separation to obtain a supernatant; removing free protein and pigment from the supernatant with HP20 macroporous resin at a mass ratio of 1:20, and repeating for 3 times; dialyzing with clear water and redistilled water (cutoff Mw 8,000), concentrating, and freeze-drying to obtain Phellinus Baumii polysaccharide (PV-F). The calculated yield was 4.6% by weight, and the protein content was about 0.28% by elemental analysis, which was lower and similar to example 2. The chemical structure is identified to be the same as the poplar yellow polysaccharide PV-F prepared in the example 2, and the content of protein impurities is close. The yield is much lower in this example than in example 2, indicating that sufficient freezing can improve the yield.
Comparative example 1
Taking 100g of poplar yellow fruit bodies, crushing the poplar yellow fruit bodies to 2 meshes, soaking the poplar yellow fruit bodies in normal saline for 24 hours, centrifuging the poplar yellow fruit bodies, and collecting residues; leaching the residue with 1.5L water at 120 deg.C for half an hour, centrifuging, collecting residue, and repeating twice; dissolving the residue in 2L 1.25M NaOH solution, leaching twice at room temperature for 8 hr each time, neutralizing with acetic acid, and collecting supernatant; removing protein from the supernatant by a Sevag method, and repeating the steps for three times; decolorizing with 30% hydrogen peroxide, concentrating, dialyzing with clear water and distilled water (cutoff Mw is 8,000), concentrating, and freeze drying to obtain alkali extracted Phellinus linteus polysaccharide (PV-B). The yield calculated by weighing is 4.7%, and is slightly lower than that of example 1 in which freeze thawing is repeated three times, which indicates that the extraction rate of the freeze thawing method can be greatly improved. The protein content detected by Kjeldahl method is about 1.3%, and the protein content is slightly higher. The chemical structure is identified to be the same as the poplar yellow polysaccharide (PV-F) prepared in the example 1, and the content of protein impurities is slightly higher.
Example 7
The tumor cells selected were cervical tumor cells (Hela) and liver cancer cells HepG 2. The cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 100U/mL of penicillin and 100. mu.g/mL of streptomycin. The cells were cultured in an incubator (37 ℃ C., 5% CO 2). Cell viability was determined using the MTT method. After cell resuspension, 6000 cells per well were seeded in 96-well plates and placed in an incubator (37 ℃, 5% CO 2). After 24 hours of incubation, the wells were washed twice with PBS and 200 μ L of fresh medium was added to each well, 20 μ L of samples with different concentrations were added to each well of the experimental group, the incubation was performed for 48 hours, 20 μ L of MTT (5mg/mL) solution was added to each well, the incubation was continued in an incubator for 4 hours, the supernatant was discarded, 150 μ L of DMSO was added to each well, and after the formazan crystals were dissolved, the optical intensity (OD) value at 570nm was measured using an automatic microplate reader (Bio-Rad, Model 550, USA) for colorimetric detection of each well. Cell viability was calculated by (OD570, sample/OD570, control). times.100%, OD570, sample being the light intensity of each experimental group to which the sample was added, OD570, control being the light intensity of the control group (PBS). After two tumor cells are respectively treated by the polysaccharide of the poplar yellow with different concentrations for 48 hours, the survival rate of the tumor cells is shown in figure 3 and figure 4, and the inhibition effect of the two polysaccharides on the proliferation of the tumor cells is linearly enhanced within the concentration range of 2-100 mug/mL. The inhibition rate of PV-B on HeLa cells is slightly lower than that of PV-F, and the inhibition rate on HepG2 cells is slightly higher than that of PV-F, which shows that the polysaccharides extracted by the two methods have certain inhibition effect on the proliferation of tumor cell strains.
Example 8
The inflammatory response results of the polysaccharides extracted by different methods were characterized by the release of the inflammatory factor NO. NO was detected with a Griess kit (Biyuntian Biotechnology Co., Ltd., Shanghai). The cells were cultured in the same manner as in example 7 using RAW264.7, and the RAW264.7 cells were seeded in a 96-well plate and incubated for 24 hours. After PBS washing, fresh DMEM medium was added, and PBS was added as a blank control, LPS (100ng/mL) as a positive control, and different concentrations of Phellinus linteus polysaccharide PV-F, PV-B as in example 2 were added. After an additional 24 hours of incubation, the supernatant (50. mu.L) and standards were added to a 96-well plate, and 50. mu.L of room temperature Griess Reagent I and 50. mu.L of Griess Reagent II were added to each well in sequence. Absorbance was measured at 540nm using an automatic microplate reader. And (4) drawing a standard curve by the standard substance, and calculating the NO release amount of each sample and the positive control. As shown in FIG. 5, it is evident that the alkaline-extracting polysaccharide PV-B in comparative example 1 can obviously cause the inflammatory reaction of macrophages, while the polysaccharide PV-F in example 1 has a maximum concentration of 100 μ g/mL, the NO content in the culture medium is close to that of the blank group, NO inflammatory reaction is caused, and the biocompatibility is good.
In conclusion, the invention provides an extraction method of phellinus igniarius polysaccharides, which is characterized in that microwave quick thawing is carried out after repeated freezing for many times, sporocarp of phellinus igniarius is subjected to wall breaking, the yield of phellinus igniarius polysaccharides is improved, the obtained phellinus igniarius polysaccharides have obvious activity of inhibiting tumor cell proliferation, and compared with polysaccharides extracted and purified by a chemical method, the phellinus igniarius polysaccharides have good biocompatibility and do not bring inflammatory reaction, and the phellinus igniarius polysaccharides can be used in antitumor drugs or health care products.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. The method for extracting the phellinus igniarius polysaccharides from the poplar is characterized by comprising the following steps of:
(1) crushing phellinus igniarius sporocarp to 1-5 meshes, soaking the phellinus igniarius sporocarp in normal saline for 8-24 hours, centrifuging and collecting residues;
(2) boiling the phellinus igniarius sporocarp residues in boiling water at the temperature of 110-130 ℃ for 20-40 min, centrifuging and collecting residues;
(3) adding water into the residue in the step (2), freezing for 24-36h at-15-10 ℃, and then quickly thawing by microwave with the power of 3000W;
(4) adding water into the unfrozen mixture obtained in the step (3) to obtain a mixed solution, adding a complex enzyme into the mixed solution, carrying out enzymolysis for 2-4 h at 50-60 ℃, and carrying out centrifugal separation to obtain a supernatant; the complex enzyme comprises cellulase and chitinase; wherein the mass ratio of the cellulase to the chitinase is 1: 1-3; the adding amount of the complex enzyme is 0.2-0.5 ten thousandth of the weight of the mixed solution;
(5) and adsorbing the supernatant by using resin, dialyzing, concentrating and freeze-drying to obtain the phellinus igniarius polysaccharides of the poplar.
2. The method for extracting polysaccharides of Phellinus linteus of claim 1, wherein the amount of the Phellinus linteus fruiting body residue and the boiling water added in step (2) is 60-80 g/L.
3. The method for extracting phellinus linteus polysaccharides of poplar as claimed in claim 1, wherein the addition amount of the residue and the water in step (3) is 200 g/L.
4. The method for extracting phellinus linteus polysaccharides of poplar as claimed in claim 1, wherein the addition amount of the thawing mixture and the water in step (4) is 60-80 g/L.
5. The method for extracting phellinus linteus polysaccharides of poplar as claimed in claim 1, wherein the resin in step (5) is macroporous resin; wherein the mass ratio of the macroporous resin to the supernatant is 1: 20-50.
6. The use of Phellinus aspergillum polysaccharide as claimed in any one of claims 1-5 in preparing antitumor drugs or health products.
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