CN113398153B - Method for utilizing phellinus igniarius mycelium - Google Patents
Method for utilizing phellinus igniarius mycelium Download PDFInfo
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- 241000123113 Phellinus igniarius Species 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000000284 extract Substances 0.000 claims abstract description 33
- 229930003944 flavone Natural products 0.000 claims abstract description 26
- 235000011949 flavones Nutrition 0.000 claims abstract description 26
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 claims abstract description 24
- 150000002212 flavone derivatives Chemical class 0.000 claims abstract description 24
- 150000004676 glycans Chemical class 0.000 claims abstract description 24
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 24
- 239000005017 polysaccharide Substances 0.000 claims abstract description 24
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 21
- 239000007788 liquid Substances 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 68
- 239000000843 powder Substances 0.000 claims description 33
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- 241000001727 Tropicoporus linteus Species 0.000 claims description 26
- 238000002137 ultrasound extraction Methods 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000002244 precipitate Substances 0.000 claims description 19
- 238000010298 pulverizing process Methods 0.000 claims description 17
- 239000011347 resin Substances 0.000 claims description 15
- 229920005989 resin Polymers 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- WSOLOQCBIJCQIQ-LURJTMIESA-N COCCOC([C@H](CC1)NC1=O)=O Chemical compound COCCOC([C@H](CC1)NC1=O)=O WSOLOQCBIJCQIQ-LURJTMIESA-N 0.000 claims description 12
- 238000001291 vacuum drying Methods 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 9
- 238000000498 ball milling Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- -1 ethylene glycol monomethyl ether pyroglutamic acid ester Chemical class 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 claims description 5
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 3
- ODHCTXKNWHHXJC-GSVOUGTGSA-N Pyroglutamic acid Natural products OC(=O)[C@H]1CCC(=O)N1 ODHCTXKNWHHXJC-GSVOUGTGSA-N 0.000 claims description 3
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 3
- 238000005292 vacuum distillation Methods 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 abstract description 4
- 230000004151 fermentation Effects 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 abstract description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 abstract description 3
- 239000008103 glucose Substances 0.000 abstract description 3
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 abstract description 3
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 abstract description 3
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 abstract description 3
- 235000005493 rutin Nutrition 0.000 abstract description 3
- 229960004555 rutoside Drugs 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 description 8
- 229930003935 flavonoid Natural products 0.000 description 6
- 150000002215 flavonoids Chemical class 0.000 description 6
- 235000017173 flavonoids Nutrition 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 238000004821 distillation Methods 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 239000003480 eluent Substances 0.000 description 4
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000002213 flavones Chemical class 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000003809 water extraction Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical class CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 241000218213 Morus <angiosperm> Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/24—Oxygen or sulfur atoms
- C07D207/26—2-Pyrrolidones
- C07D207/273—2-Pyrrolidones with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to other ring carbon atoms
- C07D207/277—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D207/28—2-Pyrrolidone-5- carboxylic acids; Functional derivatives thereof, e.g. esters, nitriles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
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- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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Abstract
The invention discloses a method for utilizing phellinus igniarius mycelia, which relates to the technical field of biological extraction, wherein the phellinus igniarius mycelia prepared by a liquid fermentation technology are used as extraction raw materials, and the phellinus igniarius mycelia have the characteristics of high yield and stable quality, and can solve the problems of low yield and unstable quality of wild phellinus igniarius mycelia and artificially cultured phellinus igniarius mycelia; and polysaccharide extract and flavone extract are obtained through extraction, separation and purification, wherein the polysaccharide content reaches more than 38% (calculated by glucose), and the total flavone content reaches more than 55% (calculated by rutin), so that the polysaccharide extract and the flavone extract have higher market competitiveness and application value.
Description
The technical field is as follows:
the invention relates to the technical field of biological extraction, in particular to a method for utilizing phellinus igniarius mycelium.
Background art:
phellinus linteus is a fungus used as crude drug for many years, and is named because the fungus usually grows on Morus plants in the Central and south areas of China and the fruit body is yellow brown. Modern researches show that phellinus igniarius has good biological activities of resisting tumor, oxidation and aging, reducing blood sugar and the like, and active ingredients of the phellinus igniarius mainly comprise polysaccharide, terpenoid, flavonoid, sterol and the like. By using liquid fermentation technology, a large amount of Phellinus linteus mycelia can be obtained in a short period, and active metabolites thereof can be obtained therefrom. Under the conditions of wild resource shortage and great artificial cultivation difficulty, the method for obtaining the phellinus igniarius active metabolite by fermentation is an effective way for solving the problem of phellinus igniarius sporophore resource shortage, meeting market requirements and improving social and economic benefits, and has very important significance.
Since polysaccharides and flavones belong to the main components of phellinus linteus mycelium and polysaccharides and flavones have various biological activities, the present invention aims to provide a method for utilizing phellinus linteus mycelium, which is expected to ultimately obtain high-content polysaccharide products and flavone products and improve the economic benefits of phellinus linteus mycelium.
The invention content is as follows:
the invention aims to provide a method for utilizing phellinus igniarius mycelium, which extracts high-content polysaccharide and flavonoid bioactive components from the phellinus igniarius mycelium, thereby realizing the high-efficiency utilization of the phellinus igniarius mycelium.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
a method for utilizing Phellinus linteus mycelium comprises the following steps:
(1) removing impurities from Phellinus linteus mycelium, drying, and pulverizing to obtain Phellinus linteus mycelium dry powder;
(2) adding ethylene glycol monomethyl ether pyroglutamate into Phellinus linteus mycelium dry powder, performing ball milling treatment, adding hot water, performing ultrasonic extraction for 2-3 times, filtering, and mixing filtrates to obtain extract I;
(3) concentrating the extracting solution I under reduced pressure, dripping ethanol into the obtained concentrated solution for precipitation, standing, and separating to obtain precipitate and supernatant;
(4) drying the obtained precipitate, and pulverizing to obtain polysaccharide extract;
(5) distilling the obtained supernatant under reduced pressure to recover ethanol, and filtering to obtain extractive solution II;
(6) purifying the extract II with resin column, eluting with ethanol water solution after column chromatography until no flavone is detected, collecting ethanol eluate, recovering ethanol by vacuum distillation, adding ethyl acetate for extraction, separating, concentrating the upper layer liquid under reduced pressure, drying, and pulverizing to obtain flavone extract.
The grain diameter of the phellinus igniarius mycelium dry powder is 60-120 meshes.
The dosage of the ethylene glycol monomethyl ether pyroglutamic acid ester is 5-8% of the mass of the phellinus igniarius mycelium dry powder.
The ethylene glycol monomethyl ether pyroglutamic acid ester is prepared by the reaction of pyroglutamic acid and ethylene glycol monomethyl ether.
The synthetic route of the ethylene glycol monomethyl ether pyroglutamic acid ester is as follows:
structure watchAnd (4) data characterization:1H NMR(DMSO-d6,400MHz)δ:7.78(s,1H),4.14(t,2H),3.98(t,1H),3.61(t,2H),3.43(s,3H),2.56-2.18(m,4H)。
the ethylene glycol monomethyl ether pyroglutamic acid ester is a colorless liquid and is fully contacted with the phellinus igniarius mycelium dry powder through ball milling, so that on one hand, the surface energy of the powder can be reduced, the spreadability of water on the powder is enhanced, the uniform dispersion of the powder in the water is promoted, and the water extraction efficiency is improved; on the other hand, the cell wall of the phellinus igniarius mycelium can be penetrated, and the osmotic pressure of the cell wall is increased during water extraction, so that the leaching of effective components is facilitated.
The amount of the hot water is 5-20 times of the mass of the phellinus igniarius mycelium dry powder, and the temperature of the hot water is 60-80 ℃.
The power of the ultrasonic extraction is 100-1000W, and the frequency is 20-60 kHz.
The ultrasonic extraction temperature is 60-80 deg.C, and the time is 2-8 h. The ultrasonic extraction method has the advantages of high extraction efficiency, short extraction time and low extraction temperature.
Macroporous adsorption resin is selected to fill a resin column, and the selective adsorption of the macroporous adsorption resin is utilized to separate and purify the flavone.
The volume concentration of the ethanol water solution is 60-80%. The characteristic that flavone is insoluble in water but easily soluble in ethanol is utilized.
The drying is vacuum drying or freeze drying. Vacuum drying and freeze drying belong to low-temperature drying, and the problem that the structure of the bioactive components is damaged by high-temperature drying is avoided.
The flow rate of the upper column is 0.5-2BV/h, and the flow rate of the elution is 0.5-2 BV/h. The separation time is shortened and the separation effect is improved by controlling the flow rate of the upper column and the flow rate of elution.
The method adopts water as an extraction solvent and ethylene glycol monomethyl ether pyroglutamate as an extraction aid to realize the high-efficiency extraction of polysaccharide and flavonoid bioactive components from phellinus igniarius mycelium, has mild extraction conditions and simple and convenient operation, and solves the problems that the active structure of polysaccharide is damaged and the extract needs to be neutralized by the existing alkaline leaching method and the problems of high cost, long extraction time and harsh extraction conditions of the enzymatic hydrolysis extraction method.
The invention has the beneficial effects that: according to the invention, the phellinus igniarius mycelium prepared by using a liquid fermentation technology is used as an extraction raw material, and the phellinus igniarius mycelium has the characteristics of high yield and stable quality, so that the problems of low yield and unstable quality of wild phellinus igniarius mycelium and artificially cultivated phellinus igniarius mycelium can be solved; and polysaccharide extract and flavone extract are obtained through extraction, separation and purification, wherein the polysaccharide content reaches more than 38% (calculated by glucose), and the total flavone content reaches more than 55% (calculated by rutin), so that the polysaccharide extract and the flavone extract have higher market competitiveness and application value.
The specific implementation mode is as follows:
in order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
The ethylene glycol monomethyl ether pyroglutamate in the following examples is synthesized by the following steps:
adding thionyl chloride (47.6g, 0.4mol) into pyroglutamic acid (12.9g, 0.1mol), heating to 60 ℃ for reaction for 3h, and distilling under reduced pressure to remove excessive thionyl chloride; adding ethylene glycol monomethyl ether (8.0g, 0.105mol) and triethylamine (10.1g, 0.1mol), heating to 60 deg.C, reacting for 2h, naturally cooling to 25 deg.C, adding 250mL ethyl acetate and 250mL water, extracting, standing, collecting the upper layer liquid, and distilling under reduced pressure to remove ethyl acetate to obtain ethylene glycol monomethyl ether pyroglutamate.
Example 1
(1) Removing impurities from Phellinus linteus mycelium, freeze drying, and pulverizing to obtain Phellinus linteus mycelium dry powder sieved with 100 mesh sieve.
(2) Adding 5% of ethylene glycol monomethyl ether pyroglutamate in an amount which is 5 times of the mass of the phellinus igniarius mycelium dry powder into 100g of phellinus igniarius mycelium dry powder, performing ball milling for 2 hours, adding 10 times of hot water in an amount which is 10 times of the mass of the phellinus igniarius mycelium dry powder, performing ultrasonic extraction for 2 times, wherein the temperature of the hot water is 60 ℃, the power of the ultrasonic extraction is 500W, the frequency is 40kHz, the temperature of the ultrasonic extraction is 60 ℃, filtering, and combining the filtrate to obtain an extracting solution I.
(3) Concentrating the extractive solution I under reduced pressure to 1/3, adding ethanol dropwise into the obtained concentrated solution for precipitation, stopping adding ethanol dropwise until no precipitate is separated out, standing for 3 hr, and separating to obtain precipitate and supernatant.
(4) Vacuum drying the precipitate, and pulverizing to obtain polysaccharide extract.
(5) Distilling the obtained supernatant under reduced pressure to recover ethanol, and filtering to obtain extractive solution II.
(6) And (3) purifying the extracting solution II by using a resin column (macroporous adsorption resin D101), eluting the extracting solution II by using an ethanol water solution with the volume concentration of 60% after the extracting solution II is loaded on the column until no flavone is detected, wherein the flow rate of the loading on the column is 1BV/h, and the elution flow rate is 2BV/h, collecting ethanol eluent, recovering ethanol through reduced pressure distillation, respectively adding 150mL of ethyl acetate to extract for 2 times, separating liquid, concentrating the upper layer liquid under reduced pressure, then performing vacuum drying, and crushing to obtain the flavone extract.
Example 2
(1) Removing impurities from Phellinus linteus mycelium, freeze drying, and pulverizing to obtain Phellinus linteus mycelium dry powder sieved with 100 mesh sieve.
(2) Adding ethylene glycol monomethyl ether pyroglutamate 8% times the mass of the phellinus igniarius mycelium dry powder into 100g of phellinus igniarius mycelium dry powder, carrying out ball milling treatment for 2h, adding hot water 15 times the mass of the phellinus igniarius mycelium dry powder, carrying out ultrasonic extraction for 3 times, wherein the extraction time is 2h, the temperature of the hot water is 80 ℃, the power of the ultrasonic extraction is 500W, the frequency is 40kHz, the temperature of the ultrasonic extraction is 80 ℃, filtering, and combining the filtrate to obtain an extracting solution I.
(3) Concentrating the extractive solution I under reduced pressure to 1/4, adding ethanol dropwise into the obtained concentrated solution for precipitation, stopping adding ethanol dropwise until no precipitate is separated out, standing for 3 hr, and separating to obtain precipitate and supernatant.
(4) Vacuum drying the precipitate, and pulverizing to obtain polysaccharide extract.
(5) Distilling the obtained supernatant under reduced pressure to recover ethanol, and filtering to obtain extractive solution II.
(6) Purifying the extract II with a resin column (macroporous adsorption resin AB-8), eluting with 70 vol% ethanol water solution after column loading until no flavone is detected, wherein the flow rate of column loading is 1BV/h and the elution flow rate is 2BV/h, collecting ethanol eluate, recovering ethanol through reduced pressure distillation, respectively adding 200mL ethyl acetate to extract for 2 times, separating liquid, concentrating the upper layer liquid under reduced pressure, vacuum drying, and pulverizing to obtain the flavone extract.
Example 3
(1) Removing impurities from Phellinus linteus mycelium, freeze drying, and pulverizing to obtain Phellinus linteus mycelium dry powder sieved with 100 mesh sieve.
(2) Adding ethylene glycol monomethyl ether pyroglutamate 8% times the mass of the phellinus igniarius mycelium dry powder into 100g of phellinus igniarius mycelium dry powder, performing ball milling for 2h, adding hot water 12 times the mass of the phellinus igniarius mycelium dry powder, performing ultrasonic extraction for 3 times, wherein the hot water temperature is 70 ℃, the ultrasonic extraction power is 500W, the frequency is 40kHz, the ultrasonic extraction temperature is 70 ℃, filtering, and combining the filtrates to obtain an extracting solution I.
(3) Concentrating the extractive solution I under reduced pressure to 1/4, adding ethanol dropwise into the obtained concentrated solution for precipitation, stopping adding ethanol dropwise until no precipitate is separated out, standing for 3 hr, and separating to obtain precipitate and supernatant.
(4) Vacuum drying the precipitate, and pulverizing to obtain polysaccharide extract.
(5) Distilling the obtained supernatant under reduced pressure to recover ethanol, and filtering to obtain extractive solution II.
(6) And (3) purifying the extracting solution II by using a resin column (macroporous adsorption resin DM301), eluting the extracting solution II by using an ethanol water solution with the volume concentration of 65% after the extracting solution II is loaded on the column until no flavone is detected, wherein the flow rate of the loading on the column is 2BV/h, and the elution flow rate is 2BV/h, collecting ethanol eluent, recovering ethanol through reduced pressure distillation, respectively adding 150mL of ethyl acetate to extract for 2 times, separating liquid, concentrating the upper layer liquid under reduced pressure, then performing vacuum drying, and crushing to obtain the flavone extract.
Comparative example 1
The same as in example 3 was repeated except that ethylene glycol monomethyl ether pyroglutamate in example 3 was replaced with ethylene glycol monomethyl ether.
(1) Removing impurities from Phellinus linteus mycelium, freeze drying, and pulverizing to obtain Phellinus linteus mycelium dry powder sieved with 100 mesh sieve.
(2) Adding ethylene glycol monomethyl ether 8% times the mass of the phellinus igniarius mycelium dry powder into 100g of phellinus igniarius mycelium dry powder, performing ball milling for 2 hours, adding hot water 12 times the mass of the phellinus igniarius mycelium dry powder, performing ultrasonic extraction for 3 times, wherein the hot water temperature is 70 ℃, the ultrasonic extraction power is 500W, the frequency is 40kHz, the ultrasonic extraction temperature is 70 ℃, filtering, and combining the filtrate to obtain an extracting solution I.
(3) Concentrating the extractive solution I under reduced pressure to 1/4, adding ethanol dropwise into the obtained concentrated solution for precipitation, stopping adding ethanol dropwise until no precipitate is separated out, standing for 3 hr, and separating to obtain precipitate and supernatant.
(4) Vacuum drying the precipitate, and pulverizing to obtain polysaccharide extract.
(5) Distilling the obtained supernatant under reduced pressure to recover ethanol, and filtering to obtain extractive solution II.
(6) And (3) purifying the extracting solution II by using a resin column (macroporous adsorption resin DM301), eluting the extracting solution II by using an ethanol water solution with the volume concentration of 65% after the extracting solution II is loaded on the column until no flavone is detected, wherein the flow rate of the loading on the column is 2BV/h, and the elution flow rate is 2BV/h, collecting ethanol eluent, recovering ethanol through reduced pressure distillation, respectively adding 150mL of ethyl acetate to extract for 2 times, separating liquid, concentrating the upper layer liquid under reduced pressure, then performing vacuum drying, and crushing to obtain the flavone extract.
Comparative example 2
The procedure of example 3 was repeated except that ethylene glycol monomethyl ether pyroglutamate in example 3 was removed.
(1) Removing impurities from Phellinus linteus mycelium, freeze drying, and pulverizing to obtain Phellinus linteus mycelium dry powder sieved with 100 mesh sieve.
(2) Ball-milling 100g of Phellinus linteus mycelium dry powder for 2h, adding hot water with the mass 12 times of that of Phellinus linteus mycelium dry powder, performing ultrasonic extraction for 3 times, each time for 2.5h, wherein the hot water temperature is 70 deg.C, the ultrasonic extraction power is 500W, the frequency is 40kHz, and the ultrasonic extraction temperature is 70 deg.C, filtering, and mixing filtrates to obtain extract I.
(3) Concentrating the extractive solution I under reduced pressure to 1/4, adding ethanol dropwise into the obtained concentrated solution for precipitation, stopping adding ethanol dropwise until no precipitate is separated out, standing for 3 hr, and separating to obtain precipitate and supernatant.
(4) Vacuum drying the precipitate, and pulverizing to obtain polysaccharide extract.
(5) And distilling the obtained supernatant under reduced pressure to recover ethanol, and filtering to obtain an extracting solution II.
(6) And (3) purifying the extracting solution II by using a resin column (macroporous adsorption resin DM301), eluting the extracting solution II by using an ethanol water solution with the volume concentration of 65% until no flavone is detected, wherein the flow rate of the column is 2BV/h, and the elution flow rate is 2BV/h, collecting ethanol eluent, recovering ethanol by reduced pressure distillation, respectively adding 150mL of ethyl acetate to extract for 2 times, separating liquid, concentrating the upper layer liquid under reduced pressure, drying in vacuum, and crushing to obtain the flavone extract.
The contents of polysaccharides and total flavonoids in the polysaccharide extracts and the flavone extracts prepared in examples 1 to 3 and comparative examples 1 to 2 were measured, respectively, and the results are shown in Table 1.
TABLE 1
| Polysaccharide content (measured as glucose) | Total Flavonoids content (in terms of rutin) | |
| Example 1 | 38.4 | 55.2 |
| Example 2 | 39.5 | 57.3 |
| Example 3 | 40.7 | 59.4 |
| Comparative example 1 | 32.1 | 46.8 |
| Comparative example 2 | 30.6 | 44.5 |
As can be seen from the data in Table 1, the addition of ethylene glycol monomethyl ether pyroglutamate during the extraction process is beneficial to increasing the content of polysaccharides and total flavonoids in the extract, i.e., the ethylene glycol monomethyl ether pyroglutamate can act as an extraction aid.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (3)
1. A method for utilizing phellinus igniarius mycelium is characterized by comprising the following steps: the method comprises the following steps:
(1) removing impurities from Phellinus linteus mycelium, drying, and pulverizing to obtain Phellinus linteus mycelium dry powder;
(2) adding ethylene glycol monomethyl ether pyroglutamate into Phellinus linteus mycelium dry powder, performing ball milling treatment, adding hot water, performing ultrasonic extraction for 2-3 times, filtering, and mixing filtrates to obtain extract I;
(3) concentrating the extracting solution I under reduced pressure, dripping ethanol into the obtained concentrated solution for precipitation, standing, and separating to obtain precipitate and supernatant;
(4) drying the obtained precipitate, and pulverizing to obtain polysaccharide extract;
(5) distilling the obtained supernatant under reduced pressure to recover ethanol, and filtering to obtain extractive solution II;
(6) purifying the extract II with resin column, eluting with ethanol water solution after loading onto the column until no flavone is detected, collecting ethanol eluate, recovering ethanol by vacuum distillation, adding ethyl acetate for extraction, separating, concentrating the upper layer liquid under reduced pressure, drying, and pulverizing to obtain flavone extract;
the dosage of the ethylene glycol monomethyl ether pyroglutamic acid ester is 5-8% of the mass of the phellinus igniarius mycelium dry powder;
the ethylene glycol monomethyl ether pyroglutamic acid ester is prepared by the reaction of pyroglutamic acid and ethylene glycol monomethyl ether;
the amount of the hot water is 5-20 times of the mass of the phellinus igniarius mycelium dry powder, and the temperature of the hot water is 60-80 ℃;
the power of the ultrasonic extraction is 100-1000W, and the frequency is 20-60 kHz;
the ultrasonic extraction temperature is 60-80 ℃, and the time is 2-8 h;
the volume concentration of the ethanol water solution is 60-80%;
the flow rate of the upper column is 0.5-2BV/h, and the flow rate of the elution is 0.5-2 BV/h.
2. The method for utilizing Phellinus linteus mycelium according to claim 1, wherein: the grain diameter of the phellinus igniarius mycelium dry powder is 60-120 meshes.
3. The method for utilizing Phellinus linteus mycelium according to claim 1, wherein: the drying is vacuum drying or freeze drying.
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| CN104829742A (en) * | 2015-05-14 | 2015-08-12 | 江苏大学 | Phellinus linteus polysaccharide separation and purification method |
| CN104910289A (en) * | 2015-06-16 | 2015-09-16 | 江苏大学 | Method for continuously preparing phellinus igniarius mycelium polysaccharide |
| CN106491662A (en) * | 2016-11-03 | 2017-03-15 | 淳安千岛湖桑都食用菌专业合作社 | A kind of method for cultivating extraction polysaccharide and flavones simultaneously in waste material from Phellinus |
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| CN104829742A (en) * | 2015-05-14 | 2015-08-12 | 江苏大学 | Phellinus linteus polysaccharide separation and purification method |
| CN104910289A (en) * | 2015-06-16 | 2015-09-16 | 江苏大学 | Method for continuously preparing phellinus igniarius mycelium polysaccharide |
| CN106491662A (en) * | 2016-11-03 | 2017-03-15 | 淳安千岛湖桑都食用菌专业合作社 | A kind of method for cultivating extraction polysaccharide and flavones simultaneously in waste material from Phellinus |
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