CN114716502A - Method for extracting phellinus linteus triterpenoid - Google Patents
Method for extracting phellinus linteus triterpenoid Download PDFInfo
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- CN114716502A CN114716502A CN202210394757.8A CN202210394757A CN114716502A CN 114716502 A CN114716502 A CN 114716502A CN 202210394757 A CN202210394757 A CN 202210394757A CN 114716502 A CN114716502 A CN 114716502A
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- 241000001727 Tropicoporus linteus Species 0.000 title claims abstract description 76
- XBZYWSMVVKYHQN-MYPRUECHSA-N (4as,6as,6br,8ar,9r,10s,12ar,12br,14bs)-10-hydroxy-2,2,6a,6b,9,12a-hexamethyl-9-[(sulfooxy)methyl]-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid Chemical compound C1C[C@H](O)[C@@](C)(COS(O)(=O)=O)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C XBZYWSMVVKYHQN-MYPRUECHSA-N 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000000605 extraction Methods 0.000 claims abstract description 67
- 150000003648 triterpenes Chemical class 0.000 claims abstract description 40
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 38
- 230000005684 electric field Effects 0.000 claims abstract description 30
- 238000002386 leaching Methods 0.000 claims abstract description 23
- 239000000843 powder Substances 0.000 claims abstract description 19
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 12
- 239000002904 solvent Substances 0.000 claims description 40
- 230000005496 eutectics Effects 0.000 claims description 38
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 37
- 238000002156 mixing Methods 0.000 claims description 34
- 239000012074 organic phase Substances 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000007864 aqueous solution Substances 0.000 claims description 23
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 229940088598 enzyme Drugs 0.000 claims description 20
- 239000008367 deionised water Substances 0.000 claims description 17
- 229910021641 deionized water Inorganic materials 0.000 claims description 17
- 241000123113 Phellinus igniarius Species 0.000 claims description 14
- 239000000706 filtrate Substances 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 14
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 13
- 238000001704 evaporation Methods 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 235000011837 pasties Nutrition 0.000 claims description 8
- 238000007873 sieving Methods 0.000 claims description 8
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 7
- 229930182821 L-proline Natural products 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 7
- 235000014655 lactic acid Nutrition 0.000 claims description 7
- 239000004310 lactic acid Substances 0.000 claims description 7
- 239000012071 phase Substances 0.000 claims description 7
- 229960002429 proline Drugs 0.000 claims description 7
- 238000010298 pulverizing process Methods 0.000 claims description 7
- 108010059892 Cellulase Proteins 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 6
- 108010059820 Polygalacturonase Proteins 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 6
- 229940106157 cellulase Drugs 0.000 claims description 6
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 229940055729 papain Drugs 0.000 claims description 6
- 235000019834 papain Nutrition 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 4
- 238000007710 freezing Methods 0.000 abstract description 2
- 230000008014 freezing Effects 0.000 abstract description 2
- 239000000284 extract Substances 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- -1 triterpene compound Chemical class 0.000 description 2
- 241000222382 Agaricomycotina Species 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010012741 Diarrhoea haemorrhagic Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010027514 Metrorrhagia Diseases 0.000 description 1
- 206010038084 Rectocele Diseases 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 231100000540 amenorrhea Toxicity 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J75/00—Processes for the preparation of steroids in general
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a method for extracting phellinus linteus triterpenes, which comprises the following steps: preparing phellinus linteus powder, freezing and crushing at low temperature, leaching, carrying out enzymolysis in a high-voltage pulse alternating electric field, carrying out primary rotary evaporation, extracting, and carrying out secondary rotary evaporation to finally obtain the phellinus linteus triterpenoid with high purity. The method extracts the phellinus linteus triterpenoids to the maximum extent by two times of ethanol ultrasonic extraction, and then obtains the high-purity phellinus linteus triterpenoids by the combined extraction of high-voltage pulse electric field treatment and enzymolysis method and secondary rotary evaporation. The method provided by the invention has the advantages of high extraction rate, short extraction time and convenient operation, and the obtained phellinus linteus triterpenes have high purity and great medicinal value.
Description
Technical Field
The invention belongs to the technical field of active substance extraction, and particularly relates to a method for extracting phellinus linteus triterpenes.
Background
Phellinus igniarius (Pellinus igniarius) belongs to medical fungi of Hymenomycetes of Basidiomycotina (Bas. Idiomycotina), has the reputation of forest gold, has 7 scientific names used as Phellinus igniarius in the literature, has more than 42 Chinese names, is sweet and pungent in taste and nontoxic, and is used for treating metrorrhagia, bloody stranguria, rectocele and bloody diarrhea, leukorrhagia and amenorrhea. Modern researches show that the phellinus igniarius has various pharmacological activities of resisting tumor, oxidation and inflammation, reducing blood sugar, resisting gastric ulcer, resisting bacteria and the like. Especially has wide prospect in the aspect of preventing and treating tumors, has become a hot point of research of various researchers, and is hopeful to be developed into a new anti-tumor medicament. Triterpenoids encompass not less than hundreds of compound components, and phellinus linteus triterpenes are one of the main components of phellinus linteus, and have many functions such as antiviral, immunity enhancement and hepatic fibrosis resistance.
In the existing research, the supercritical CO exists in the triterpenoid2The method comprises a plurality of extraction methods such as an extraction method, an ultrasonic-assisted method, a microwave method, a reflux extraction method and the like, but in the process of extracting the phellinus linteus triterpenoid, when separation adsorbents or extraction raw materials such as ethanol and the like are carried and separated by the extraction raw materials, the loss of the phellinus linteus triterpenoid is easily caused, the extraction amount of the phellinus linteus triterpenoid is reduced, and the phellinus linteus raw materials are not screened before preparation, so that the purity of the later-stage preparation of the phellinus linteus triterpenoid cannot be guaranteed, the quality of the phellinus linteus triterpenoid is reduced, and the medicinal value of the phellinus linteus triterpenoid is reduced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the method for extracting the phellinus linteus triterpenes, the method has the advantages of high extraction rate, short extraction time and convenient operation, and the obtained phellinus linteus triterpenes have high purity and great medicinal value.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for extracting phellinus linteus triterpenes comprises the following steps:
(1) freeze-drying and crushing at low temperature: pulverizing cleaned Phellinus Linteus mycelium at low temperature, and sieving to obtain Phellinus Linteus powder;
(2) leaching: adding the phellinus linteus powder obtained in the step (1) into a eutectic solvent aqueous solution, performing ultrasonic extraction, centrifuging after extraction is finished, and taking supernate to obtain a leaching liquor and extraction residues;
(3) enzymolysis in a high-voltage pulse alternating electric field: adding the extraction residue obtained in the step (2) into deionized water, then adding a complex enzyme, treating through a high-voltage pulse alternating electric field, and filtering to obtain an enzymolysis filtrate and filter residue;
(4) primary rotary evaporation: mixing the first leaching solution obtained in the step (2) and the enzymolysis filtrate obtained in the step (3), filtering, performing rotary evaporation, and evaporating to obtain paste substances with quality not reduced any more, thereby obtaining crude triterpenoids of phellinus igniarius;
(5) and (3) extraction: adding the crude phellinus linteus triterpenoids obtained in the step (4) into a rotary evaporation bottle, then adding deionized water and n-butanol, and then extracting with a separating funnel to leave a first organic phase; adding the remaining water phase into the separating funnel again, adding n-butyl alcohol, and performing secondary extraction to obtain a second organic phase;
(6) secondary rotary evaporation: and (4) mixing the first organic phase and the second organic phase in the step (5), performing rotary evaporation until the mass of the paste-like substance is not reduced any more, and drying to obtain the phellinus linteus triterpenoid.
Preferably, the crushing temperature in the step (1) is-65 to-45 ℃, and the screening mesh number is 40 to 80 meshes.
Preferably, the low eutectic solvent in the eutectic solvent aqueous solution in the step (2) is prepared by mixing L-proline and lactic acid in a ratio of substance amount of 1: 1, mixing and heating at 80 ℃ for 1 h; the volume concentration of the low eutectic solvent in the eutectic solvent water solution is 75 percent; the feed-liquid ratio of the phellinus igniarius powder to the eutectic solvent aqueous solution is 1 g: 25-30 mL.
Preferably, in the step (2), the ultrasonic extraction temperature is 20-30 ℃, the ultrasonic extraction time is 15-30 min, the centrifugation speed is 4000-5000 r/min, and the centrifugation time is 15-30 min.
Preferably, the complex enzyme in the step (3) is prepared by mixing pectinase, cellulase and papain, and the mass ratio of the three enzymes is 1: 1: 1; the mass ratio of the extraction residues to the complex enzyme is 10: 1.
preferably, in the step (3), the electric field intensity in the high-voltage pulse alternating electric field treatment chamber is 20-30 kv/cm, the number of pulses is 12-14, and the extracted residue liquid enters the high-voltage pulse electric field extraction device through a pump for extraction.
Preferably, the high-voltage pulse alternating electric field treatment in step (3) means that a sine-wave alternating current is instantaneously turned off by a solid-state relay and a continuous alternating current is changed into a pulse current, and the duty ratio thereof is controlled by a PLC.
Preferably, in the step (4), the rotary evaporation temperature is 55-65 ℃, and the rotation speed is 80-120 r/min.
Preferably, in the step (5), the volume ratio of the n-butanol to the deionized water is 1: 1, the feed-liquid ratio of the crude phellinus linteus triterpene to a mixed solution of n-butanol and deionization is 1 g: 25-30 mL.
Preferably, the rotary evaporation temperature in the step (6) is 70-80 ℃, and the rotary speed is 60-80 r/min.
Preferably, the drying temperature in the step (6) is 60-80 ℃, and the drying time is 2-4 h.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method comprises the following steps of firstly, freezing and crushing phellinus igniarius mycelia at a low temperature, wherein cell sap in cell walls can be in a frozen state at the low temperature, and the cell sap cannot remain on the inner wall of a machine during crushing; meanwhile, the phellinus igniarius hyphae in a low-temperature state are high in brittleness and easy to crush, and the crushing efficiency is effectively improved; the crushed phellinus linteus powder is processed at a lower temperature, is immediately put into a eutectic solvent aqueous solution at the normal temperature, and under the action of thermal expansion, cooling and permeation, cell walls of phellinus linteus mycelia are damaged, and triterpenoids are extracted more quickly.
(2) The invention combines the high-voltage pulse electric field treatment and the enzymolysis method for extraction, and the high-frequency current directly acts on the biological molecules to excite the molecular activity, so that the effective components are quickly dissolved out. When the high-frequency current with alternating positive and negative passes through the raw material soaked by the solvent, the tissue structure of the raw material is loosened to form an effective component dissolving channel. When current passes through the raw material particles in the solution, the whole cells in the raw material are instantaneously heated and expanded to be destroyed due to the existence of the resistor, and the internal substances are dissolved by the solvent. Meanwhile, due to the action of current, the enzymolysis speed is accelerated, the dissolution of the triterpenoid is further improved, and the rapid, continuous, normal-temperature and efficient enhanced extraction effect is formed.
(3) The method purifies the crude triterpenoids of the phellinus linteus by two-time extraction and two-time rotary evaporation, so that the obtained phellinus linteus triterpenoids have high purity and great medicinal value.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
A method for extracting phellinus linteus triterpenes comprises the following steps:
(1) freeze-drying and crushing at low temperature: pulverizing cleaned Phellinus Linteus mycelium at-65 deg.C, and sieving with 40 mesh sieve to obtain Phellinus Linteus powder;
(2) leaching: adding the phellinus linteus powder (10g) obtained in the step (1) into a eutectic solvent aqueous solution (250mL), performing ultrasonic extraction for 15min at 25 ℃, and centrifuging at the rotating speed of 4500r/min for 15min after extraction is finished to obtain a leaching solution and extraction residues; the low eutectic solvent in the eutectic solvent aqueous solution is prepared by mixing L-proline and lactic acid according to the mass ratio of 1: 1, mixing and heating at 80 ℃ for 1 h; the volume concentration of the low eutectic solvent in the eutectic solvent aqueous solution is 75 percent;
(3) enzymolysis in a high-voltage pulse alternating electric field: adding 10g of the extraction residue obtained in the step (2) into deionized water, then adding 1g of complex enzyme, treating through a high-voltage pulse alternating electric field, and filtering to obtain enzymolysis filtrate and filter residue; the compound enzyme is prepared by mixing pectinase, cellulase and papain, wherein the mass ratio of the three enzymes is 1: 1: 1; the electric field intensity in the high-voltage pulse alternating electric field treatment chamber is 20kv/cm, and the number of pulses is 12;
(4) primary rotary evaporation: mixing the leaching liquor obtained in the step (2) and the enzymolysis filtrate obtained in the step (3), filtering, performing rotary evaporation at 55 ℃, wherein the rotation speed is 80r/min, and evaporating until the mass is not reduced any more to obtain crude phellinus linteus triterpene;
(5) adding the crude phellinus linteus triterpene (10g) obtained in the step (4) into a rotary evaporation bottle, then adding 125mL of deionized water and 125mL of n-butanol, and then extracting with a separating funnel to leave a first organic phase; adding the remaining water phase into the separating funnel again, adding 125mL of n-butanol, and performing secondary extraction to obtain a second organic phase;
(6) and (4) mixing the first organic phase and the second organic phase in the step (5), performing rotary evaporation at 70 ℃, wherein the rotation speed is 60r/min, evaporating until the mass of the pasty substance is not reduced any more, and drying at 60 ℃ for 4h to obtain the phellinus linteus triterpenoid.
Example 2
A method for extracting phellinus linteus triterpenes comprises the following steps:
(1) freeze-drying and crushing at low temperature: pulverizing cleaned Phellinus Linteus mycelium at-55 deg.C, and sieving with 60 mesh sieve to obtain Phellinus Linteus powder;
(2) leaching: adding the phellinus igniarius powder (10g) obtained in the step (1) into a eutectic solvent aqueous solution (250mL), performing ultrasonic extraction for 20min at 25 ℃, and centrifuging for 20min at the rotating speed of 4500r/min after extraction is finished to obtain a leaching liquor and extraction residues; the low eutectic solvent in the eutectic solvent aqueous solution is prepared by mixing L-proline and lactic acid according to the mass ratio of 1: 1, mixing and heating at 80 ℃ for 1 h; the volume concentration of the low eutectic solvent in the eutectic solvent aqueous solution is 75 percent;
(3) enzymolysis in a high-voltage pulse alternating electric field: adding 10g of the extraction residue obtained in the step (2) into deionized water, then adding 1g of complex enzyme, treating through a high-voltage pulse alternating electric field, and filtering to obtain enzymolysis filtrate and filter residue; the compound enzyme is prepared by mixing pectinase, cellulase and papain, wherein the mass ratio of the three enzymes is 1: 1: 1; the electric field intensity in the high-voltage pulse alternating electric field treatment chamber is 25kv/cm, and the number of pulses is 13;
(4) primary rotary evaporation: mixing the leaching liquor obtained in the step (2) and the enzymolysis filtrate obtained in the step (3), filtering, performing rotary evaporation at 60 ℃, wherein the rotation speed is 100r/min, and evaporating until the mass is not reduced any more to obtain crude phellinus linteus triterpene;
(5) adding the crude phellinus linteus triterpene (10g) obtained in the step (4) into a rotary evaporation bottle, then adding 125mL of deionized water and 125mL of n-butanol, and then extracting with a separating funnel to leave a first organic phase; adding the remaining water phase into the separating funnel again, adding 125mL of n-butanol, and performing secondary extraction to obtain a second organic phase;
(6) and (3) mixing the first organic phase and the second organic phase in the step (5), performing rotary evaporation at 75 ℃, wherein the rotation speed is 70r/min, evaporating until the mass of the pasty substance is not reduced any more, and drying at 70 ℃ for 3h to obtain the phellinus linteus triterpenoid.
Example 3
A method for extracting phellinus linteus triterpenes comprises the following steps:
(1) freeze-drying and crushing at low temperature: pulverizing cleaned Phellinus Linteus mycelium at-45 deg.C, and sieving with 80 mesh sieve to obtain Phellinus Linteus powder;
(2) leaching: adding the phellinus igniarius powder (10g) obtained in the step (1) into a eutectic solvent aqueous solution (300mL), performing ultrasonic extraction for 30min at 30 ℃, and centrifuging for 30min at the rotating speed of 5000r/min after extraction is finished to obtain a leaching solution and extraction residues; the low eutectic solvent in the eutectic solvent aqueous solution is prepared by mixing L-proline and lactic acid according to the mass ratio of 1: 1, mixing and heating at 80 ℃ for 1 h; the volume concentration of the low eutectic solvent in the eutectic solvent aqueous solution is 75 percent;
(3) enzymolysis in a high-voltage pulse alternating electric field: adding 10g of the extraction residue obtained in the step (2) into deionized water, then adding 1g of complex enzyme, treating through a high-voltage pulse alternating electric field, and filtering to obtain enzymolysis filtrate and filter residue; the compound enzyme is prepared by mixing pectinase, cellulase and papain, wherein the mass ratio of the three enzymes is 1: 1: 1; the electric field intensity in the high-voltage pulse alternating electric field treatment chamber is 30kv/cm, and the number of pulses is 14;
(4) primary rotary evaporation: mixing the leaching liquor obtained in the step (2) and the enzymolysis filtrate obtained in the step (3), filtering, performing rotary evaporation at 65 ℃, wherein the rotating speed is 120r/min, and evaporating until the mass is not reduced any more to obtain a pasty substance, namely the crude triterpenoid of phellinus igniarius;
(5) adding the crude phellinus linteus triterpene (10g) obtained in the step (4) into a rotary evaporation bottle, then adding 125mL of deionized water and 125mL of n-butanol, and then extracting with a separating funnel to leave a first organic phase; adding the remaining water phase into the separating funnel again, adding 125mL of n-butanol, and performing secondary extraction to obtain a second organic phase;
(6) and (4) mixing the first organic phase and the second organic phase in the step (5), performing rotary evaporation at 80 ℃, wherein the rotation speed is 80r/min, evaporating until the mass of the pasty substance is not reduced any more, and drying at 80 ℃ for 2h to obtain the phellinus linteus triterpenoid.
Comparative example 1
A method for extracting phellinus linteus triterpenes comprises the following steps:
(1) freeze-drying and crushing at low temperature: pulverizing cleaned Phellinus Linteus mycelium at-65 deg.C, and sieving with 40 mesh sieve to obtain Phellinus Linteus powder;
(2) leaching: adding the phellinus linteus powder (10g) obtained in the step (1) into a eutectic solvent aqueous solution (250mL), performing ultrasonic extraction for 15min at 25 ℃, and centrifuging for 15min at the rotating speed of 4500r/min after extraction is finished to obtain a leaching liquor and extraction residues; the low eutectic solvent in the eutectic solvent aqueous solution is prepared by mixing L-proline and lactic acid according to the mass ratio of 1: 1, mixing and heating at 80 ℃ for 1 h; the volume concentration of the low eutectic solvent in the eutectic solvent aqueous solution is 75 percent;
(3) enzymolysis: adding 10g of the extraction residue obtained in the step (2) into deionized water, then adding 1g of complex enzyme, performing enzymolysis for 20min, and filtering to obtain enzymolysis filtrate and filter residue; the compound enzyme is prepared by mixing pectinase, cellulase and papain, wherein the mass ratio of the three enzymes is 1: 1: 1;
(4) primary rotary evaporation: mixing the leaching liquor obtained in the step (2) and the enzymolysis filtrate obtained in the step (3), filtering, performing rotary evaporation at 55 ℃, wherein the rotation speed is 80r/min, and evaporating until the mass is not reduced any more to obtain crude phellinus linteus triterpene;
(5) adding the crude phellinus linteus triterpene (10g) obtained in the step (4) into a rotary evaporation bottle, then adding 125mL of deionized water and 125mL of n-butanol, and then extracting with a separating funnel to leave a first organic phase; adding the remaining water phase into the separating funnel again, adding 125mL of n-butanol, and performing secondary extraction to obtain a second organic phase;
(6) and (4) mixing the first organic phase and the second organic phase in the step (5), performing rotary evaporation at 70 ℃, wherein the rotation speed is 60r/min, evaporating until the mass of the pasty substance is not reduced any more, and drying at 60 ℃ for 4h to obtain the phellinus linteus triterpenoid.
Comparative example 2
A method for extracting phellinus linteus triterpenes comprises the following steps:
(1) freeze-drying and crushing at low temperature: pulverizing cleaned Phellinus Linteus mycelium at-65 deg.C, and sieving with 40 mesh sieve to obtain Phellinus Linteus powder;
(2) leaching: adding the phellinus linteus powder (10g) obtained in the step (1) into a eutectic solvent aqueous solution (250mL), performing ultrasonic extraction for 15min at 25 ℃, and centrifuging for 15min at the rotating speed of 4500r/min after extraction is finished to obtain a leaching liquor and extraction residues; the low eutectic solvent in the eutectic solvent aqueous solution is prepared by mixing L-proline and lactic acid according to the mass ratio of 1: 1, mixing and heating at 80 ℃ for 1 h; the volume concentration of the low eutectic solvent in the eutectic solvent aqueous solution is 75 percent;
(3) treating in a high-voltage pulse alternating electric field: adding 10g of the extraction residue obtained in the step (2) into deionized water, treating by a high-voltage pulse alternating electric field, and filtering to obtain filtrate and filter residue; the electric field intensity in the high-voltage pulse alternating electric field treatment chamber is 20kv/cm, and the number of pulses is 12;
(4) primary rotary evaporation: mixing the leaching liquor obtained in the step (2) and the filtrate obtained in the step (3), filtering, performing rotary evaporation at 55 ℃, wherein the rotation speed is 80r/min, and evaporating until the mass is not reduced any more to obtain a pasty substance, namely the crude triterpenoid of phellinus linteus;
(5) adding the crude phellinus linteus triterpene (10g) obtained in the step (4) into a rotary evaporation bottle, then adding 125mL of deionized water and 125mL of n-butanol, and then extracting with a separating funnel to leave a first organic phase; adding the remaining water phase into the separating funnel again, adding 125mL of n-butanol, and performing secondary extraction to obtain a second organic phase;
(6) and (4) mixing the first organic phase and the second organic phase in the step (5), performing rotary evaporation at 70 ℃, wherein the rotation speed is 60r/min, evaporating until the mass of the pasty substance is not reduced any more, and drying at 60 ℃ for 4h to obtain the phellinus linteus triterpenoid.
The extraction rate and the purity of the triterpenoid in the examples 1 to 3 and the comparative examples 1 to 2 were calculated respectively, the calculation formula is as follows, and the calculation results are summarized in table 1.
The leaching rate (g/g) is the crude triterpenoid quantity of phellinus igniarius/phellinus igniarius powder quantity multiplied by 100 percent;
the purity of the triterpenoid (g/g) is equal to the total triterpene amount/crude triterpene amount of phellinus igniarius x 100%.
Wherein the triterpenoid is measured by ultraviolet spectrophotometry, and the type of the spectrophotometer is UV-1800 type produced by Shanghai precision instruments and meters.
TABLE 1 triterpene compound extraction yield and purity test results
| Extraction ratio of crude triterpene from Phellinus linteus (%) | Triterpene Compound purity (%) | |
| Example 1 | 14.9 | 76 |
| Example 2 | 16.5 | 83 |
| Example 3 | 15.6 | 78 |
| Comparative example 1 | 7.2 | 53 |
| Comparative example 2 | 8.0 | 59 |
As shown in the above results, the extraction rate of crude triterpenoids from Phellinus linteus in comparative examples 1-2 is lower than that in examples 1-3, which shows that the extraction rate of triterpenoids from Phellinus linteus can be improved by the extraction method of the present invention, and the purity of triterpenoids from Phellinus linteus in examples 1-3 is higher than that in comparative examples 1-2, which shows that the purity of triterpenoids from Phellinus linteus is improved by the purification method of the present invention.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. The method for extracting the phellinus linteus triterpenes is characterized by comprising the following steps:
(1) freeze-drying and crushing at low temperature: pulverizing cleaned Phellinus Linteus mycelium at low temperature, and sieving to obtain Phellinus Linteus powder;
(2) leaching: adding the phellinus linteus powder obtained in the step (1) into a eutectic solvent aqueous solution, performing ultrasonic extraction, centrifuging after extraction is finished, and taking supernate to obtain a leaching liquor and extraction residues;
(3) enzymolysis in a high-voltage pulse alternating electric field: adding the extraction residue obtained in the step (2) into deionized water, then adding a complex enzyme, treating through a high-voltage pulse alternating electric field, and filtering to obtain an enzymolysis filtrate and filter residue;
(4) primary rotary evaporation: mixing the first leaching liquor obtained in the step (2) and the enzymolysis filtrate obtained in the step (3), filtering, performing rotary evaporation, and evaporating until the mass of the pasty substance is not reduced any more, thereby obtaining crude triterpenoids of phellinus linteus;
(5) and (3) extraction: adding the crude phellinus linteus triterpenoids obtained in the step (4) into a rotary evaporation bottle, then adding deionized water and n-butanol, and then extracting with a separating funnel to leave a first organic phase; adding the remaining water phase into the separating funnel again, adding n-butanol, and performing secondary extraction to obtain a second organic phase;
(6) secondary rotary evaporation: and (4) mixing the first organic phase and the second organic phase in the step (5), performing rotary evaporation until the mass of the paste-like substance is not reduced any more, and drying to obtain the phellinus linteus triterpenoid.
2. The extraction method according to claim 1, wherein the crushing temperature in the step (1) is-65 to-45 ℃, and the sieving mesh number is 40 to 80 meshes.
3. The extraction method according to claim 1, wherein the low eutectic solvent in the eutectic solvent aqueous solution in the step (2) is prepared by mixing L-proline and lactic acid in a ratio of 1: 1, mixing and heating at 80 ℃ for 1 h; the volume concentration of the low eutectic solvent in the eutectic solvent aqueous solution is 75 percent; the feed-liquid ratio of the phellinus igniarius powder to the eutectic solvent aqueous solution is 1 g: 25-30 mL.
4. The extraction method according to claim 1, wherein the ultrasonic extraction temperature in step (2) is 20-30 ℃, the ultrasonic extraction time is 15-30 min, the centrifugation rate is 4000-5000 r/min, and the centrifugation time is 15-30 min.
5. The extraction method according to claim 1, wherein the complex enzyme in the step (3) is prepared by mixing pectinase, cellulase and papain, and the mass ratio of the three enzymes is 1: 1: 1; the mass ratio of the extraction residues to the complex enzyme is 10: 1.
6. the extraction method according to claim 1, wherein in the step (3), the electric field intensity in the high-voltage pulse alternating electric field treatment chamber is 20-30 kv/cm, the number of pulses is 12-14, and the extraction residue liquid enters the high-voltage pulse electric field extraction device through a pump for extraction.
7. The extraction method according to claim 1, wherein in the step (4), the rotary evaporation temperature is 55-65 ℃ and the rotation speed is 80-120 r/min.
8. The extraction process according to claim 1, wherein in the step (5), the volume ratio of n-butanol to deionized water is 1: 1, the feed-liquid ratio of the crude phellinus linteus triterpene to a mixed solution of n-butanol and deionized water is 1 g: 25-30 mL.
9. The extraction method according to claim 1, wherein the rotary evaporation temperature in step (6) is 70-80 ℃ and the rotary speed is 60-80 r/min.
10. The extraction method according to claim 1, wherein the drying temperature in the step (6) is 60 to 80 ℃ and the drying time is 2 to 4 hours.
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