A method for extracting mulberry linterus polysaccharide
Through the soaking and temperature treatment of specific ethanol solution, combined with alcohol precipitation and column chromatography purification technology, the problem of low extraction rate of mulberry yellow polysaccharides is solved, and efficient and stable polysaccharide extraction is achieved, which improves the yield and purity of polysaccharides. Significant medicinal value.
Patent Information
- Application Number
- CN202310533272.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-12
- Publication Date
- 2024-12-27
- Estimated Expiration
- 2043-05-12
AI Technical Summary
In the existing mulberry yellow polysaccharide extraction methods, the polysaccharide yield is low and the extraction time is unstable, resulting in incomplete extraction of polysaccharides or increased energy consumption, and poor stability of polysaccharide substances.
Immersion and temperature treatment are used to improve the yield of polysaccharides. Impairment and column chromatography purification technology are used to remove impurities to improve the purification and yield of polysaccharides. Ionized substances are adsorbed by DEAE-52 cellulose column chromatography. Remove the coarseness and extract the essence.
The extraction rate and purity of mulberry yellow polysaccharides are improved, the extraction time is short and the operation is easy, and the obtained polysaccharides have high medicinal value.
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of edible and medicinal fungus polysaccharides, and particularly relates to a method for extracting mulberry linterus polysaccharides. Background Art
[0002] Phellinus igniarius is a perennial macrofungus with considerable medicinal value. It is named because it grows mostly on mulberry plants and its fruiting bodies are mostly yellow-brown. It is also known as firewood phyllopore, mulberry, and mulberry ear. Phellinus igniarius has complex ingredients and contains multiple active ingredients such as polysaccharides, flavonoids, triterpenes, sterols, and coumarin compounds. In my country, mulberry igniarius is used to treat metrorrhagia, hemorrhage, rectal bleeding, umbilical and abdominal pain, amenorrhea, and spleen deficiency diarrhea. Modern pharmacological studies have shown that mulberry igniarius polysaccharides have anti-tumor, liver fibrosis inhibition, immunity improvement, blood sugar reduction, and antiviral effects, and their anti-tumor activity is better than that of various fungal drugs. Therefore, in recent years, more and more people have paid attention to it, and the research on mulberry mulberry igniarius polysaccharides has gradually increased. In the prior art, ultrasonic extraction is usually used for the extraction of mulberry mulberry igniarius polysaccharides. The above method has the problem of low polysaccharide yield, and its extraction time is unstable. The extraction time is too short, resulting in incomplete polysaccharide extraction. The extraction time is too long, which will not only increase energy consumption, but also deteriorate the stability of polysaccharide substances, thereby reducing the polysaccharide yield. Therefore, providing a method for extracting mulberry linterus polysaccharide and improving the yield of mulberry linterus polysaccharide are problems that technicians in this field need to solve urgently. Summary of the invention
[0003] In view of the problem that the polysaccharide yield extracted by the existing extraction method is low, the present invention provides a method for extracting mulberry linterus polysaccharide.
[0004] In order to solve the above technical problems, the technical solution provided by the present invention is:
[0005] The present invention provides a method for extracting mulberry linterus polysaccharide, comprising the following steps:
[0006] Step a, crushing the Phellinus linteus fruiting bodies, adding them to an ethanol solution, soaking, solid-liquid separation, drying, crushing, and sieving to obtain Phellinus linteus powder;
[0007] Step b, adding the mulberry powder to water, soaking at 95°C-100°C, filtering, concentrating the obtained filtrate, adding 62%-67% ethanol solution for alcohol precipitation, solid-liquid separation, drying, and obtaining mulberry linterus crude polysaccharide;
[0008] Step c, adding the crude mulberry linterus polysaccharide into water to obtain a crude mulberry linterus polysaccharide solution, subjecting the crude mulberry linterus polysaccharide solution to polyamide column chromatography, eluting with deionized water, collecting the eluate, concentrating, and drying to obtain primary mulberry linterus polysaccharide;
[0009] Step d, adding the primary mulberry linterus polysaccharide into water to obtain a primary mulberry linterus polysaccharide solution, subjecting the primary mulberry linterus polysaccharide solution to DEAE-52 cellulose anion exchange column chromatography purification, eluting with deionized water, 0.08mol / mL-0.12mol / mL sodium chloride solution, 0.28mol / mL-0.32mol / mL sodium chloride solution and 0.48mol / mL-0.52mol / mL sodium chloride solution in sequence, collecting the eluate, dialyzing for desalination, concentrating and drying to obtain mulberry linterus polysaccharide.
[0010] Compared with the prior art, the extraction method of mulberry linterus polysaccharide provided by the present invention can achieve a degreasing effect by soaking mulberry linterus fruiting bodies in a specific ethanol solution, and further, soaking mulberry linterus powder at a specific temperature can improve the ability of polysaccharide to diffuse outward to the best, and effectively improve the yield of polysaccharide; by determining a specific concentration of ethanol solution for alcohol precipitation, polysaccharide can be precipitated to the greatest extent; selecting polyamide for column chromatography can improve the removal rate of protein and pigment in crude mulberry linterus polysaccharide while improving the retention rate of mulberry linterus polysaccharide; DEAE-52 cellulose is used as a filler for column chromatography purification, which can adsorb ionic substances such as protein, so as to remove the coarse and retain the fine, achieve the purpose of separation, and further improve the yield of mulberry linterus polysaccharide.
[0011] The method for extracting mulberry linterus polysaccharide provided by the invention has high extraction rate, short extraction time, convenient operation, high yield of mulberry linterus polysaccharide obtained, and great medicinal value.
[0012] Preferably, in step a, the mass-to-volume ratio of the Phellinus linteus fruiting body and the ethanol solution is 1:8-10, the unit of mass is g, and the unit of volume is mL.
[0013] Preferably, in step a, the volume concentration of the ethanol solution is 92%-95%.
[0014] Preferably, in step b, the mass-to-volume ratio of the mulberry linterus powder and water is 1:105-110, the unit of mass is g, and the unit of volume is mL.
[0015] The preferred ratio can further improve the yield of Phellinus igniarius polysaccharide.
[0016] Preferably, in step b, the mulberry linterus powder is soaked in water for extraction twice.
[0017] Preferably, in step b, the soaking time is 10h-13h.
[0018] Too long a soaking time will easily lead to a decrease in the polysaccharide yield, while too short a soaking time will lead to insufficient polysaccharide extraction. The optimal soaking time can maximize the yield of Phellinus igniarius polysaccharide.
[0019] Preferably, in step b, the alcohol precipitation time is 24h-28h.
[0020] Preferably, in step b, the temperature of the alcohol precipitation is 4°C-6°C.
[0021] Preferably, in step c, the concentration of the Phellinus igniarius crude polysaccharide solution is 100 mg / mL-110 mg / mL.
[0022] Preferably, in step c, the volume ratio of the phellinus igniarius crude polysaccharide solution to deionized water is 1-2:300-330.
[0023] Preferably, in step d, the concentration of the primary mulberry linterus polysaccharide solution is 20 mg / mL-25 mg / mL.
[0024] Preferably, in step d, the volume ratio of the primary mulberry linterus polysaccharide solution to deionized water is 2-5:150-180.
[0025] Preferably, the volume ratio of the deionized water, 0.08mol / mL-0.12mol / mL sodium chloride solution, 0.28mol / mL-0.32mol / mL sodium chloride solution and 0.48mol / mL-0.52mol / mL sodium chloride solution is 1-1.2:1-1.2:1-1.2:1-1.2.
[0026] The preferred eluent and its dosage can further increase the yield of polysaccharide.
[0027] Preferably, in step d, the molecular weight cut-off during dialysis desalination is 3500Da-3700Da.
[0028] The preferred molecular weight cut-off can remove sodium chloride and reduce the content of impurities in mulberry linterus polysaccharide.
[0029] Preferably, in step d, the dialysis desalination time is 24h-28h.
[0030] Preferably, in step d, collecting the eluate is collecting the eluate after elution with 0.08 mol / mL-0.12 mol / mL sodium chloride solution. DETAILED DESCRIPTION
[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0032] Example 1
[0033] This embodiment provides a method for extracting mulberry linterus polysaccharide, comprising the following steps:
[0034] Step a, crushing the fruiting bodies of Phellinus linteus and soaking them in a 95% ethanol solution, filtering, drying, and crushing them through an 80-mesh sieve, retaining the sieve undersize, to obtain Phellinus linteus powder, wherein the mass-to-volume ratio of the fruiting bodies of Phellinus linteus and the ethanol solution is 1:10, the unit of mass is g, and the unit of volume is mL;
[0035] Step b, adding the mulberry powder to water, soaking at 95° C. for 13 hours to obtain an immersion solution, repeating the immersion extraction twice, filtering the immersion solution, concentrating it, and performing alcohol precipitation in a 62% ethanol solution for 28 hours, centrifuging at 4500r / min for 15 minutes, and drying to obtain mulberry linterus crude polysaccharide, wherein the mass volume ratio of mulberry powder to water is 1:110, the unit of mass is g, and the unit of volume is mL;
[0036] Step c, adding the crude polysaccharide of Phellinus linteus to water to prepare a crude polysaccharide solution of Phellinus linteus with a concentration of 100 mg / mL, subjecting the crude polysaccharide solution of Phellinus linteus to polyamide column (4 cm*58 cm) chromatography, eluting with deionized water, collecting the eluate, concentrating, and drying to obtain primary Phellinus linteus polysaccharide, wherein the loading amount of the crude polysaccharide solution of Phellinus linteus is 1 mL, the flow rate of the crude polysaccharide solution of Phellinus linteus is 5 mL / min, the usage amount of deionized water is 300 mL, and the flow rate of deionized water is 5 mL / min;
[0037] Step d, adding the primary mulberry linterus polysaccharide to water to prepare a primary mulberry linterus polysaccharide solution with a concentration of 25 mg / mL, subjecting the primary mulberry linterus polysaccharide solution to DEAE-52 cellulose anion exchange column (55 cm*2.6 cm) chromatography purification, and eluting with deionized water, 0.1 mol / mL sodium chloride solution, 0.3 mol / mL sodium chloride solution and 0.5 mol / mL sodium chloride solution in sequence, collecting 0.1 mL of the eluted sodium chloride solution, dialyzing in a dialysis bag (molecular weight cutoff of 3500 Da) for 48 h, concentrating, and drying to obtain mulberry linterus polysaccharide, wherein the loading amount of the primary mulberry linterus polysaccharide solution is 2 mL, the flow rate of the primary mulberry linterus polysaccharide solution is 8 mL / min, the flow rates of deionized water, 0.1 mol / mL sodium chloride solution, 0.3 mol / mL sodium chloride solution and 0.5 mol / mL sodium chloride solution are all 1 mL / min, and the usage amount is 180 mL.
[0038] Example 2
[0039] This embodiment provides a method for extracting mulberry linterus polysaccharide, comprising the following steps:
[0040] Step a, crushing the fruiting bodies of Phellinus linteus and soaking them in a 92% ethanol solution, filtering, drying, and crushing them through an 80-mesh sieve, retaining the sieve undersize, to obtain Phellinus linteus powder, wherein the mass-to-volume ratio of the fruiting bodies of Phellinus linteus and the ethanol solution is 1:8, the unit of mass is g, and the unit of volume is mL;
[0041] Step b, adding the mulberry powder to water, soaking it at 105° C. for 10 hours to obtain an immersion solution, repeating the immersion extraction twice, filtering the immersion solution, concentrating it, and performing alcohol precipitation in a 67% ethanol solution for 24 hours, centrifuging it at 4000 r / min for 20 minutes, and drying it to obtain mulberry linterus crude polysaccharide, wherein the mass volume ratio of mulberry powder to water is 1:105, the unit of mass is g, and the unit of volume is mL;
[0042] Step c, adding the crude mulberry linterus polysaccharide to water to prepare a crude mulberry linterus polysaccharide solution with a concentration of 110 mg / mL, subjecting the crude mulberry linterus polysaccharide solution to polyamide column (4 cm*58 cm) chromatography, eluting with deionized water, collecting the eluate, concentrating, and drying to obtain primary mulberry linterus polysaccharide, wherein the loading amount of the crude mulberry linterus polysaccharide solution is 1 mL, the flow rate of the crude mulberry linterus polysaccharide solution is 5 mL / min, the amount of deionized water used is 330 mL, and the flow rate of deionized water is 5 mL / min;
[0043] Step d, adding the primary mulberry linterus polysaccharide to water to prepare a primary mulberry linterus polysaccharide solution with a concentration of 20 mg / mL, subjecting the primary mulberry linterus polysaccharide solution to DEAE-52 cellulose anion exchange column (55 cm*2.6 cm) chromatography purification, eluting with deionized water, 0.1 mol / mL sodium chloride solution, 0.3 mol / mL sodium chloride solution and 0.5 mol / mL sodium chloride solution in sequence, collecting 0.1 mL of the eluted sodium chloride solution, dialyzing in a dialysis bag (molecular weight cutoff of 3700 Da) for 24 h, concentrating, and drying to obtain mulberry linterus polysaccharide, wherein the loading amount of the primary mulberry linterus polysaccharide solution is 5 mL, the flow rate of the primary mulberry linterus polysaccharide solution is 5 mL / min, the flow rates of deionized water, 0.1 mol / mL sodium chloride solution, 0.3 mol / mL sodium chloride solution and 0.5 mol / mL sodium chloride solution are all 1 mL / min, and the usage amount is 150 mL.
[0044] Example 3
[0045] This embodiment provides a method for extracting mulberry linterus polysaccharide, comprising the following steps:
[0046] Step a, crushing the fruiting bodies of Phellinus linteus and soaking them in a 93% ethanol solution, filtering, drying, and crushing them through an 80-mesh sieve, retaining the sieve undersize, to obtain Phellinus linteus powder, wherein the mass-to-volume ratio of the fruiting bodies of Phellinus linteus and the ethanol solution is 1:9, the unit of mass is g, and the unit of volume is mL;
[0047] Step b, adding the mulberry powder to water, soaking it at 100° C. for 12 hours to obtain an immersion solution, repeating the immersion extraction twice, filtering the immersion solution, concentrating it, and performing alcohol precipitation in a 65% ethanol solution for 26 hours, centrifuging it at 4300 r / min for 18 minutes, and drying it to obtain mulberry linterus crude polysaccharide, wherein the mass volume ratio of mulberry powder to water is 1:108, the unit of mass is g, and the unit of volume is mL;
[0048] Step c, adding the crude polysaccharide of Phellinus linteus to water to prepare a crude polysaccharide solution of Phellinus linteus with a concentration of 105 mg / mL, subjecting the crude polysaccharide solution of Phellinus linteus to polyamide column (4 cm*58 cm) chromatography, eluting with deionized water, collecting the eluate, concentrating, and drying to obtain primary Phellinus linteus polysaccharide, wherein the loading amount of the crude polysaccharide solution of Phellinus linteus is 1.5 mL, the flow rate of the crude polysaccharide solution of Phellinus linteus is 6 mL / min, the usage amount of deionized water is 320 mL, and the flow rate of deionized water is 6 mL / min;
[0049] Step d, adding the primary mulberry linterus polysaccharide to water to prepare a primary mulberry linterus polysaccharide solution with a concentration of 22 mg / mL, subjecting the primary mulberry linterus polysaccharide solution to DEAE-52 cellulose anion exchange column (55 cm*2.6 cm) chromatography purification, eluting with deionized water, 0.1 mol / mL sodium chloride solution, 0.3 mol / mL sodium chloride solution and 0.5 mol / mL sodium chloride solution in sequence, collecting 0.1 mL of the eluted sodium chloride solution, dialyzing in a dialysis bag (molecular weight cutoff of 3600 Da) for 25 h, concentrating, and drying to obtain mulberry linterus polysaccharide, wherein the loading amount of the primary mulberry linterus polysaccharide solution is 3 mL, the flow rate of the primary mulberry linterus polysaccharide solution is 6 mL / min, the flow rates of deionized water, 0.1 mol / mL sodium chloride solution, 0.3 mol / mL sodium chloride solution and 0.5 mol / mL sodium chloride solution are all 1 mL / min, and the usage amount is 160 mL.
[0050] Comparative Example 1
[0051] This comparative example provides a method for extracting mulberry linterus polysaccharide. The difference between this comparative example and Example 1 is that polyamide is replaced by AB-8 macroporous resin, and the other operating steps are the same as those in Example 1.
[0052] Comparative Example 2
[0053] This comparative example provides a method for extracting mulberry linterus polysaccharide. The difference between this comparative example and Example 1 is that in step d, DEAE-52 cellulose is replaced by DEAE Sepharose, and the other operating steps are the same as those in Example 1.
[0054] The yield and purity of mulberry linterus polysaccharide extracted in Examples 1-3 and Comparative Examples 1-2 were calculated respectively, wherein the purified polysaccharide yield % (g / g) = (purified polysaccharide mass / crude polysaccharide mass before purification) × 100%; the crude polysaccharide content, purified polysaccharide mass, crude polysaccharide mass before purification and polysaccharide purity were all tested by phenol-sulfuric acid method; the specific test results are shown in Table 1:
[0055] Table 1
[0056] Test items Yield of purified polysaccharide from Phellinus igniarius / % Purification of Phellinus igniarius polysaccharide / % Example 1 58.15％ 67.92％ Example 2 59.24％ 71.75％ Example 3 60.79％ 69.03％ Comparative Example 1 50.24％ 53.24％ Comparative Example 2 48.32％ 58.12％
[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for extracting mulberry linterus polysaccharide, characterized in that: The steps include: Step a, crushing the Phellinus linteus fruiting bodies, adding them to an ethanol solution, soaking, solid-liquid separation, drying, crushing, and sieving to obtain Phellinus linteus powder; Step b, adding the mulberry powder to water, soaking at 95°C-100°C, filtering, concentrating the obtained filtrate, adding 62%-67% ethanol solution for alcohol precipitation, solid-liquid separation, and drying to obtain mulberry linterus crude polysaccharide; Step c, adding the crude mulberry linterus polysaccharide into water to obtain a crude mulberry linterus polysaccharide solution, subjecting the crude mulberry linterus polysaccharide solution to polyamide column chromatography, eluting with deionized water, collecting the eluate, concentrating, and drying to obtain primary mulberry linterus polysaccharide; Step d, adding the primary mulberry linterus polysaccharide into water to obtain a primary mulberry linterus polysaccharide solution, subjecting the primary mulberry linterus polysaccharide solution to DEAE-52 cellulose anion exchange column chromatography purification, eluting with deionized water, 0.08mol / mL-0.12mol / mL sodium chloride solution, 0.28mol / mL-0.32mol / mL sodium chloride solution and 0.48mol / mL-0.52mol / mL sodium chloride solution in sequence, collecting the eluate after elution with 0.08mol / mL-0.12mol / mL sodium chloride solution, dialyzing for desalination, concentrating, and drying to obtain mulberry linterus polysaccharide; In step a, the mass-to-volume ratio of the Phellinus linteus fruiting body and the ethanol solution is 1:8-10, the unit of mass is g, and the unit of volume is mL; In step b, the soaking time is 10h-13h; In step b, the alcohol precipitation time is 24h-28h.
2. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step a, the volume concentration of the ethanol solution is 92%-95%.
3. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step b, the mass volume ratio of the mulberry linterus powder and water is 1:105-110, the unit of mass is g, and the unit of volume is mL.
4. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step c, the concentration of the Phellinus igniarius crude polysaccharide solution is 100 mg / mL-110 mg / mL.
5. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step c, the volume ratio of the phellinus linterus crude polysaccharide solution to deionized water is 1-2:300-330.
6. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step d, the concentration of the primary mulberry linterus polysaccharide solution is 20 mg / mL-25 mg / mL.
7. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step d, the volume ratio of the primary mulberry linterus polysaccharide solution to deionized water is 2-5:150-180.
8. The method for extracting phellinus linteus polysaccharide according to claim 1, characterized in that: In step d, the volume ratio of the deionized water, 0.08mol / mL-0.12mol / mL sodium chloride solution, 0.28mol / mL-0.32mol / mL sodium chloride solution and 0.48mol / mL-0.52mol / mL sodium chloride solution is 1-1.2:1-1.2:1-1.2:1-1.
2.
9. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step d, the molecular weight cut-off during dialysis desalination is 3500Da-3700Da.
10. The method for extracting phellin polysaccharide according to claim 1, characterized in that: In step d, the dialysis desalination time is 24h-28h.