CN112321663A - Method for simultaneously purifying and extracting steroid and total triterpene from phellinus igniarius - Google Patents

Method for simultaneously purifying and extracting steroid and total triterpene from phellinus igniarius Download PDF

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CN112321663A
CN112321663A CN202011130975.8A CN202011130975A CN112321663A CN 112321663 A CN112321663 A CN 112321663A CN 202011130975 A CN202011130975 A CN 202011130975A CN 112321663 A CN112321663 A CN 112321663A
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steroid
phellinus igniarius
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Abstract

The invention discloses a method for simultaneously purifying and extracting steroid and total triterpene from phellinus igniarius, which adopts the processes of complex enzyme enzymolysis, porous covalent organic framework material COF-701 enrichment and multi-frequency ultrasonic auxiliary extraction, and realizes the simultaneous high-efficiency purification and enrichment of steroid and total triterpene in phellinus igniarius by optimizing parameters such as sample liquid, eluent, ultrasonic frequency and the like. The method has the advantages of good stability, high selectivity, large adsorption capacity, simplicity and controllability in operation and the like, and can be used for purifying and enriching steroid and total triterpenes in various complex samples.

Description

Method for simultaneously purifying and extracting steroid and total triterpene from phellinus igniarius
Technical Field
The invention relates to a method for simultaneously purifying and extracting steroid and total triterpene in phellinus igniarius, belonging to the field of natural organic chemistry.
Background
Phellinus igniarius is a common name of a traditional rare medicinal fungus in China, and has various pharmacological effects such as tumor resistance, oxidation resistance, immunity improvement and the like. The natural fruiting body of Phellinus Linteus contains abundant high molecular polysaccharides, triterpenes, proteins, minerals, amino acids, sterols, etc. The triterpenoid has the effects of regulating immunity, inhibiting cancer cells, regulating blood pressure, protecting liver, reducing blood fat, resisting allergy and inflammation and the like, and the pharmacological action and the clinical effect of the triterpenoid are well established. Steroid compounds are derived from cholesterol, and have anti-inflammatory, in vivo sodium/potassium ratio regulating, blood glucose level regulating, sexual function regulating, and secondary sexual maturation promoting effects. At present, more researches are carried out on the extraction, separation and purification of phellinus igniarius polysaccharides, and less researches are carried out on steroids and triterpenoids.
Disclosure of Invention
The technical task of the invention is to make up the defects of the prior art, and provide the method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
(1) drying the Phellinus linteus subjected to impurity removal at 60 ℃, crushing, and sieving with a 40-mesh sieve to obtain Phellinus linteus dry powder;
(2) adding a disodium hydrogen phosphate buffer solution into the dry phellinus igniarius powder, uniformly mixing, adding a complex enzyme according to 0.2-1.0% of the mass of the dry phellinus igniarius powder, performing enzymolysis for 30-90 minutes at 45-55 ℃, and performing centrifugal separation on an enzymolysis liquid to obtain a supernatant and a precipitate;
(3) adding a covalent organic framework material COF-701 into the supernatant in the step (2), wherein the addition amount of the covalent organic framework material COF-701 is 1% of the mass of the phellinus igniarius dry powder, performing multi-frequency ultrasonic extraction, and performing centrifugal separation to obtain a COF-701 enrichment material;
(4) placing the COF-701 enrichment material into a centrifugal tube, adding an eluent, carrying out ultrasonic oscillation for 1 minute, and carrying out centrifugal separation to obtain a supernatant and COF-701; and concentrating the supernatant by a rotary evaporator under reduced pressure to obtain the steroid and the total triterpene product.
The disodium hydrogen phosphate buffer solution comprises disodium hydrogen phosphate and citric acid, the pH value is 4.0-6.0, and the addition amount of the disodium hydrogen phosphate buffer solution is 5-10 times of the mass of phellinus linteus dry powder.
The compound enzyme consists of cellulase and glucose oxidase, wherein the mass ratio of the cellulase to the glucose oxidase is 3: 1-1: 3.
the covalent organic framework material COF-701 is obtained by polymerizing 2,4, 6-trimethyl-1, 3, 5-triazine and 4,4' -biphenyldicarboxaldehyde at 150 ℃ under the catalysis of trifluoroacetic acid, and has a specific surface area of 1715m2g-1The pore diameter is 1.1nm, the composition and the crystallinity can be maintained under strong acid and alkaline conditions, and the structural formula is as follows:
Figure RE-GDA0002863579120000021
the multi-frequency ultrasonic extraction has the ultrasonic frequency of 20-80kHz, two-by-two combination or three simultaneous combination, the ultrasonic treatment power of 100-1000W and the extraction time of 30-300 s.
And (4) centrifuging at 700-2000 rpm for 10-30 min.
The eluent is one or two or more of ethanol, glycerol, dimethyl carbonate, limonene and ethyl acetate; the addition amount of the eluent is 2-5 times of the mass of the COF-701 enrichment material.
And the ultrasonic frequency is 25kHz, and the ultrasonic processing power is 600W.
The steroid and total triterpene products are a mixture containing Phellinane L (1 shown in figure 1), Phellinene E (2 shown in figure 1), Phellinulin D (3 shown in figure 1), Phellinulin I (4 shown in figure 1), Phellinulin K (5 shown in figure 1), beer sterol (6 shown in figure 1), beta-sitosterol (7 shown in figure 1) and perfornol (8 shown in figure 1), and the extraction rate is 60-80%.
Compared with the prior art, the method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius has the following advantages:
(1) according to the invention, a multi-frequency ultrasonic synergistic extraction method is added in the extraction of the steroid and the total triterpene of phellinus igniarius, so that the extraction time is obviously shortened, the solvent consumption is reduced, and the extraction rate is improved; the obtained steroid and total triterpene extract has high purity, does not need subsequent separation treatment, and has wide medicinal and edible application prospect.
(2) COF-701 is a highly ordered porous crystalline material composed of light elements C and N connected together, with low density and high specific surface area (1715 m)2g-1) Can stably exist under strong acid and alkaline conditions, and can be repeatedly recycled as an enrichment material; the aperture is 1.1nm, the pore channel contains a pi electron conjugated system, and the high selective adsorption performance on steroid and total triterpene compounds is realized; compared with other porous adsorption materials, the porous adsorption material has incomparable advantages.
Drawings
FIG. 1 shows the types of extracted steroid and total triterpene products, wherein FIG. 1 shows Phelline L, FIG. 1 shows Phellinene E, FIG. 2 shows Phellinene E, FIG. 1 shows Phellinin D, FIG. 1 shows 3 shows Phellinin D, FIG. 1 shows 4 shows Phellinin I, FIG. 1 shows 5 shows Phellinin K, FIG. 1 shows 6 shows beesterol, FIG. 1 shows 7 shows beta-sitosterol, and FIG. 1 shows 8 shows poriferol.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs, and the experimental methods are conventional unless otherwise specified.
The present invention will be described in detail with reference to examples.
Example 1A method for simultaneous purification and extraction of steroid and Total triterpene from Phellinus linteus
(1) Drying the Phellinus linteus subjected to impurity removal at 60 ℃, crushing, and sieving with a 40-mesh sieve to obtain Phellinus linteus dry powder;
(2) adding disodium hydrogen phosphate buffer solution into the dry powder of the phellinus igniarius, mixing uniformly, adding complex enzyme according to 0.2 percent of the mass of the dry powder of the phellinus igniarius, performing enzymolysis for 90 minutes at 55 ℃, and performing centrifugal separation on enzymolysis liquid to obtain supernatant and precipitate;
(3) adding a covalent organic framework material COF-701 into the supernatant in the step (2), wherein the addition amount of the covalent organic framework material COF-701 is 1% of the mass of the phellinus igniarius dry powder, performing multi-frequency ultrasonic extraction, and performing centrifugal separation to obtain a COF-701 enrichment material;
(4) placing the COF-701 enrichment material into a centrifugal tube, adding an eluent, carrying out ultrasonic oscillation for 1 minute, and carrying out centrifugal separation to obtain a supernatant and COF-701; and concentrating the supernatant by a rotary evaporator under reduced pressure to obtain the steroid and the total triterpene product.
The disodium hydrogen phosphate buffer solution comprises disodium hydrogen phosphate and citric acid, the pH value is 4.0, and the addition amount of the disodium hydrogen phosphate buffer solution is 5 times of the mass of phellinus igniarius dry powder;
the compound enzyme consists of cellulase and glucose oxidase, wherein the mass ratio of the cellulase to the glucose oxidase is 3: 1;
the covalent organic framework material COF-701 is obtained by polymerizing 2,4, 6-trimethyl-1, 3, 5-triazine and 4,4' -biphenyldicarboxaldehyde at 150 ℃ under the catalysis of trifluoroacetic acid, and has a specific surface area of 1715m2g-1The pore diameter is 1.1nm, the composition and the crystallinity can be maintained under the strong acid and alkaline conditions, and the structural formula of the porous materialThe following were used:
Figure RE-GDA0002863579120000031
the multi-frequency ultrasonic extraction is carried out, wherein the ultrasonic frequency is 20kHz and 80kHz, the ultrasonic processing power is 1000W, and the extraction time is 300 s;
the centrifugation is carried out for 10min at 2000 rpm;
the eluent is mixed by ethanol and limonene, and the addition amount of the eluent is 2 times of the mass of the COF-701 enrichment material; and the ultrasonic frequency is 25kHz, and the ultrasonic processing power is 600W.
Example 2A method for simultaneous purification and extraction of steroid and Total triterpene from Phellinus linteus
(1) Drying the Phellinus linteus subjected to impurity removal at 60 ℃, crushing, and sieving with a 40-mesh sieve to obtain Phellinus linteus dry powder;
(2) adding disodium hydrogen phosphate buffer solution into the dry powder of Phellinus linteus, mixing, adding complex enzyme according to 0.6% of the dry powder of Phellinus linteus, performing enzymolysis at 50 deg.C for 60 min, and centrifuging the enzymolysis solution to obtain supernatant and precipitate;
(3) adding a covalent organic framework material COF-701 into the supernatant in the step (2), wherein the addition amount of the covalent organic framework material COF-701 is 1% of the mass of the phellinus igniarius dry powder, performing multi-frequency ultrasonic extraction, and performing centrifugal separation to obtain a COF-701 enrichment material;
(4) placing the COF-701 enrichment material into a centrifugal tube, adding an eluent, carrying out ultrasonic oscillation for 1 minute, and carrying out centrifugal separation to obtain a supernatant and COF-701; and concentrating the supernatant by a rotary evaporator under reduced pressure to obtain the steroid and the total triterpene product.
The disodium hydrogen phosphate buffer solution comprises disodium hydrogen phosphate and citric acid, the pH value is 5.0, and the addition amount of the disodium hydrogen phosphate buffer solution is 7.5 times of the mass of phellinus igniarius dry powder;
the compound enzyme consists of cellulase and glucose oxidase, wherein the mass ratio of the cellulase to the glucose oxidase is 1: 1;
the covalent organic framework material COF-701 is prepared by catalyzing 2,4, 6-trimethyl-1, 3, 5-triazine and 4,4' -biphenyldicarboxaldehyde in trifluoroacetic acidPolymerized at 150 ℃ under the reaction condition, and has the specific surface area of 1715m2g-1The pore diameter is 1.1nm, the composition and the crystallinity can be maintained under strong acid and alkaline conditions, and the structural formula is as follows:
Figure RE-GDA0002863579120000041
the multi-frequency ultrasonic extraction is carried out, wherein the ultrasonic frequency is 30kHz and 70kHz, the ultrasonic processing power is 550W, and the extraction time is 135 s;
the centrifugation is carried out for 20min at 1400 rpm;
the eluent is prepared by mixing ethanol, glycerol and ethyl acetate, and the addition amount of the eluent is 3.5 times of the mass of the COF-701 enrichment material;
and the ultrasonic frequency is 25kHz, and the ultrasonic processing power is 600W.
Example 3A method for simultaneous purification and extraction of steroid and Total triterpene from Phellinus linteus
(1) Drying the Phellinus linteus subjected to impurity removal at 60 ℃, crushing, and sieving with a 40-mesh sieve to obtain Phellinus linteus dry powder;
(2) adding a disodium hydrogen phosphate buffer solution into the dry phellinus igniarius powder, uniformly mixing, adding a complex enzyme according to 1.0 percent of the mass of the dry phellinus igniarius powder, performing enzymolysis for 30 minutes at 45 ℃, and performing centrifugal separation on an enzymolysis liquid to obtain a supernatant and a precipitate;
(3) adding a covalent organic framework material COF-701 into the supernatant in the step (2), wherein the addition amount of the covalent organic framework material COF-701 is 1% of the mass of the phellinus igniarius dry powder, performing multi-frequency ultrasonic extraction, and performing centrifugal separation to obtain a COF-701 enrichment material;
(4) placing the COF-701 enrichment material into a centrifugal tube, adding an eluent, carrying out ultrasonic oscillation for 1 minute, and carrying out centrifugal separation to obtain a supernatant and COF-701; and concentrating the supernatant by a rotary evaporator under reduced pressure to obtain the steroid and the total triterpene product.
The disodium hydrogen phosphate buffer solution comprises disodium hydrogen phosphate and citric acid, the pH value is 6.0, and the addition amount of the disodium hydrogen phosphate buffer solution is 10 times of the mass of phellinus igniarius dry powder;
the compound enzyme consists of cellulase and glucose oxidase, wherein the mass ratio of the cellulase to the glucose oxidase is 1: 3;
the covalent organic framework material COF-701 is obtained by polymerizing 2,4, 6-trimethyl-1, 3, 5-triazine and 4,4' -biphenyldicarboxaldehyde at 150 ℃ under the catalysis of trifluoroacetic acid, and has a specific surface area of 1715m2g-1The pore diameter is 1.1nm, the composition and the crystallinity can be maintained under strong acid and alkaline conditions, and the structural formula is as follows:
Figure RE-GDA0002863579120000051
the multi-frequency ultrasonic extraction has the ultrasonic frequency of 20kHz,50kHz and 80kHz, the ultrasonic processing power of 100W and the extraction time of 30 s;
the centrifugation is carried out for 30min at 700 rpm;
the eluent is prepared by mixing ethanol, glycerol and dimethyl carbonate; the addition amount of the eluent is 5 times of the mass of the COF-701 enrichment material;
and the ultrasonic frequency is 25kHz, and the ultrasonic processing power is 600W.
Example 4 preparation of covalent organic framework Material COF-701
(1)0.06g of 2,4, 6-trimethyl-1, 3, 5-triazine and 0.154g of 4,4' -biphenyldialdehyde are dissolved in 4.5mL of trimethylbenzene, 4.5mL of 1, 4-dioxane, 2mL of trifluoroacetic acid and 0.25mL of acetonitrile;
(2) placing the mixed solution in a high-pressure kettle for 72 hours at the temperature of 150 ℃ to obtain a red solid;
(3) neutralizing the red solid with 0.05mol/L ammonia water solution, and washing with ethanol for 3 times;
(4) drying at 120 ℃ for 48h under vacuum to obtain yellow powder COF-701 with the yield of 85%;
the recycled covalent organic framework material COF-701 needs to be activated for 24 hours under the vacuum condition at 120 ℃.

Claims (8)

1. A method for simultaneously purifying and extracting steroid and total triterpene from phellinus igniarius is characterized by comprising the following steps:
(1) drying the Phellinus linteus subjected to impurity removal at 60 ℃, crushing, and sieving with a 40-mesh sieve to obtain Phellinus linteus dry powder;
(2) adding a disodium hydrogen phosphate buffer solution into the dry phellinus igniarius powder, uniformly mixing, adding a complex enzyme according to 0.2-1.0% of the mass of the dry phellinus igniarius powder, performing enzymolysis for 30-90 minutes at 45-55 ℃, and performing centrifugal separation on an enzymolysis liquid to obtain a supernatant and a precipitate;
(3) adding a covalent organic framework material COF-701 into the supernatant in the step (2), wherein the addition amount of the covalent organic framework material COF-701 is 1% of the mass of the phellinus igniarius dry powder, performing multi-frequency ultrasonic extraction, and performing centrifugal separation to obtain a COF-701 enrichment material;
(4) placing the COF-701 enrichment material into a centrifugal tube, adding an eluent, carrying out ultrasonic oscillation for 1 minute, and carrying out centrifugal separation to obtain a supernatant and COF-701; and concentrating the supernatant by a rotary evaporator under reduced pressure to obtain the steroid and the total triterpene product.
2. The method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius according to claim 1, which is characterized in that: the disodium hydrogen phosphate buffer solution comprises disodium hydrogen phosphate and citric acid, the pH value is 4.0-6.0, and the addition amount of the disodium hydrogen phosphate buffer solution is 5-10 times of the mass of phellinus linteus dry powder.
3. The method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius according to claim 1, which is characterized in that: the compound enzyme consists of cellulase and glucose oxidase, wherein the mass ratio of the cellulase to the glucose oxidase is 3: 1-1: 3.
4. the method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius according to claim 1, which is characterized in that: the covalent organic framework material COF-701 is obtained by polymerizing 2,4, 6-trimethyl-1, 3, 5-triazine and 4,4' -biphenyldicarboxaldehyde at 150 ℃ under the catalysis of trifluoroacetic acid, and has a specific surface area of 1715m2g-1The pore diameter is 1.1nm, the composition and the crystallinity can be maintained under strong acid and alkaline conditions, and the structural formula is as follows:
Figure FDA0002735146030000021
5. the method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius according to claim 1, which is characterized in that: the multi-frequency ultrasonic extraction has the ultrasonic frequency of 20-80kHz, two-by-two combination or three simultaneous combination, the ultrasonic treatment power of 100-1000W and the extraction time of 30-300 s.
6. The method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius according to claim 1, which is characterized in that: and (4) centrifuging at 700-2000 rpm for 10-30 min.
7. The method for simultaneously purifying and extracting the steroid and the total triterpene from the phellinus igniarius according to claim 1, which is characterized in that: the eluent is one or two or more of ethanol, glycerol, dimethyl carbonate, limonene and ethyl acetate; the addition amount of the eluent is 2-5 times of the mass of the COF-701 enrichment material.
8. The method of claim 1, wherein: and the ultrasonic frequency is 25kHz, and the ultrasonic processing power is 600W.
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CN114716502A (en) * 2022-04-15 2022-07-08 菏泽学院 Method for extracting phellinus linteus triterpenoid

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114716502A (en) * 2022-04-15 2022-07-08 菏泽学院 Method for extracting phellinus linteus triterpenoid

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