CN114990177A - Preparation method of high-purity epimedium flavone low-glycoside component - Google Patents
Preparation method of high-purity epimedium flavone low-glycoside component Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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Abstract
The invention relates to a preparation method of a high-purity epimedium flavone low-glycoside component, which can be used for industrial production. The whole preparation process comprises the following steps: extracting herba Epimedii decoction pieces with 40-70% ethanol under reflux, recovering ethanol under reduced pressure, diluting the concentrated solution with pure water, removing chlorophyll by physical method, and performing enzymolysis with cellulase to obtain herba Epimedii low sugar glycoside crude product; dissolving the epimedium low-sugar glycoside crude product with low-concentration ethanol, adsorbing the pretreated epimedium low-sugar glycoside crude product by NKA-9, ADS-17 or ADS-7 type macroporous resin, eluting the pretreated epimedium low-sugar glycoside crude product by pure water, 35-45% ethanol, 55-65% ethanol and 75-85% ethanol in sequence, collecting 55-65% and 75-85% ethanol elution parts, loading the 55-65% elution parts again for elution, combining the 75-85% ethanol elution parts, recovering ethanol under reduced pressure, and drying to obtain the epimedium low-sugar glycoside component. The technology of the invention can improve the purity of the epimedium flavone low-sugar component from 40 percent to more than 65 percent.
Description
Technical Field
The invention relates to a compound extraction method, in particular to a preparation method of a high-purity epimedium flavone low-glycoside component.
Background
The development of the raw materials of the traditional Chinese medicine preparation can be divided into three stages: the first generation of traditional Chinese medicine preparations such as pills, powder, ointment, pills and the like which take crushing, decoction and soaking as pretreatment means have the characteristics of thick and thick black, mixed release of medicine molecules and the like; the second generation of the traditional Chinese medicine preparation which takes traditional Chinese medicine extracts (powder, dry extract and fluid extract) as preparation raw materials has the defects of extensive extraction, unclear material base, disordered drug release, large oral administration dosage and the like; however, the traditional Chinese medicine preparation is developed towards the third stage, namely, high-purity components and even monomer components are used as raw materials to develop modern traditional Chinese medicine preparations with definite and controllable material bases.
Herba epimedii is a traditional Chinese medicine commonly used in clinic, and has the effects of dispelling wind-damp, tonifying kidney yang and strengthening muscles and bones, wherein the muscles and bones correspond to osteoporosis in modern medicine. A plurality of researches show that the epimedium flavonoids component is a drug effect substance which plays a role in resisting osteoporosis, and according to the quantity of glycosyl groups on 7-position of a flavone mother nucleus, the epimedium flavonoids can be divided into epimedium flavonoid polyglycoside and epimedium flavonoid oligoglycoside, wherein the epimedium flavonoid polyglycoside mainly comprises epimedin A, epimedin B, epimedin C and icariin and is a main existing form of the flavonoids component in the epimedium decoction pieces; the epimedium oligoglycoside is a secondary glycoside formed by removing glycosyl on 7-position of flavone mother nucleus from icariin, and mainly comprises baohuoside I, baohuoside II, agastachside A, agastachside B and rhamnosyl icariside II. Early-stage research shows that the epimedium polyglucoside component in epimedium decoction pieces can be converted into a corresponding epimedium low-sugar-glucoside component by in-vitro processing heating and in-vivo LPH enzyme/intestinal flora effects, experimental results of a rat in-vivo intestinal perfusion model and a Caco-2 cell model show that the epimedium low-sugar-glucoside component has better membrane permeability, and finally, the in-vivo efficacy of the epimedium low-sugar-glucoside component is found to be better by comparing the efficacies of a rat model with ovariosporulation-induced osteoporosis and a zebra fish model with prednisolone-induced osteoporosis, so that the epimedium low-sugar-glucoside component is considered to be a main component which is absorbed into blood and exerts the efficacy. In order to apply the medicine to clinic and achieve the purpose of preventing and treating diseases, the research of the medicine is finally returned to the traditional Chinese medicine preparation, so that the aim of the research is to extract and separate the epimedium herb decoction pieces, obtain a low-glucoside component of epimedium flavone with certain purity by an in vitro conversion technology, and research and develop the traditional Chinese medicine preparation by taking the low-glucoside component as a raw material.
In the prior technical scheme for preparing the epimedium flavone low-sugar-glycoside component, the invention patent of ' a method for extracting and preparing the epimedium low-sugar-glycoside component from epimedium ' (application number: 201910674431.9) is applied in the earlier stage of the subject group, and a research on preparation technology and process engineering of the epimedium low-sugar-glycoside component ' of a Master graduate paper is published, and figure 1 is an original preparation flow chart thereof. The low-sugar glycoside components obtained by column purification and separation in the case comprise baohuoside II, agastachside A, agastachside B, rhamnosyl icariside II, anhydroicaritin-3-O-rhamnose-dideoxyfuranose and baohuoside I, the purity of the obtained product is calculated to be 80% by adopting a cumulative method of relative peak areas, but the error of the method is large, and the purity is only about 40% by using absolute content calculation; due to the complex components of the Chinese herbal medicines, the resin column is often blocked in the case of column purification, so that the production cannot be carried out. In conclusion, the epimedium flavone low-sugar glycoside prepared by the technical scheme has lower component purity, can not meet the requirements of raw materials of third-generation traditional Chinese medicine preparations, and is difficult to implement in industrial production.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for preparing a high-purity epimedium flavone low-glycoside component from epimedium decoction pieces, and the method is applied to industrial production, so that the problems that the prepared epimedium flavone low-glycoside component has low purity and is difficult to industrially produce in the prior technical scheme are solved.
The technical scheme is as follows: the preparation method of the high-purity epimedium flavone low-glycoside component comprises the following steps:
(1) extracting herba Epimedii decoction pieces with ethanol under reflux, and recovering ethanol under reduced pressure to obtain concentrated solution;
(2) dissolving the concentrated solution with water for dilution, and removing chlorophyll in the solution by a physical method to obtain an enzymolysis stock solution;
(3) adding cellulase into the enzymolysis stock solution, stirring for enzymolysis, and completely reacting to obtain an enzymolysis solution;
(4) centrifuging the enzymolysis solution, removing supernatant, extracting herba Epimedii low-glycoside component in the precipitate with ethanol solution, centrifuging again to obtain supernatant, recovering ethanol under reduced pressure, and drying to obtain herba Epimedii low-glycoside component crude product;
(5) re-dissolving the coarse epimedium low-sugar component with low-concentration ethanol, fully dissolving, and then carrying out sample loading pretreatment to obtain clear epimedium low-sugar component sample loading liquid;
(6) adsorbing the sample solution with macroporous resin, sequentially eluting with water, 35-45% ethanol, 55-65% ethanol, and 75-85% ethanol for the first time, collecting 55-65% and 75-85% ethanol eluate, recovering ethanol from 55-65% ethanol eluate under reduced pressure, diluting with pure water, adsorbing with macroporous resin, and eluting again by the same method; preferably, water, 40% ethanol, 50% ethanol, 60% ethanol, 80% ethanol are adopted for the first elution;
(7) mixing the two 75-85% ethanol eluates, recovering ethanol under reduced pressure, and drying to obtain high purity epimedium low-sugar glycoside component.
The method, the ethanol solution in the step (1) is 40-70% ethanol solution. Preferably 50% ethanol solution.
The variety of the epimedium decoction pieces used in the step (1) is epimedium koreanum, epimedium cardiotrophum or epimedium hirsutum;
in the method, the concentration of the rotary solution for centrifugation in the step (2) is 0.2-0.8 g decoction pieces/mL, and the physical method for removing chlorophyll comprises centrifugation, suction filtration and sieving; if centrifugation is selected, the rotating speed is 3500-16000 rpm; if a screen is selected, the aperture of the screen is 50-120 meshes;
the method comprises the step (3) that the mass ratio of the cellulase to the epimedium decoction pieces is 1: 10-1: 50, if the epimedium koreanum is used, the mass ratio of the cellulase to the epimedium decoction pieces is 1: 10-1: 20, and when the epimedium brevicornum or the epimedium dauricum is used, the mass ratio of the cellulase to the epimedium decoction pieces is 1: 20-1: 50.
The method, the ethanol solution in the step (4) is 80-99.5% ethanol solution. Preferably a 95% ethanol solution.
According to the method, the volume fraction of ethanol in the low-concentration ethanol in the step (5) is 25-30%, and the pre-treatment method for loading comprises centrifugation and suction filtration.
According to the method, the centrifugal rotating speed during the sample loading pretreatment in the step (5) is 3500-16000 rpm.
According to the method, the macroporous resin used in the step (6) is NKA-9, ADS-17 or ADS-7 type macroporous adsorption resin, and the dosage of the macroporous resin is that: the weight ratio of the decoction pieces is 0.6: 1-1: 1;
in the method, in the step (7), the drying condition is vacuum drying, the temperature is controlled to be 65-85 ℃, and the drying time is 12-48 h.
The ethanol solution with the percentage content refers to the volume fraction of ethanol-water.
The above 40% ethanol, 50% ethanol, 60% ethanol, and 80% ethanol are ethanol-water mixture with volume fraction of 40%, 50%, 60%, and 80%.
The ingredients of the epimedium low-glycoside component are baohuoside II, agastachoside A, agastachoside B, rhamnosyl icariside II and baohuoside I, and the chemical structures of the ingredients are shown as follows, (wherein a is baohuoside I, B is baohuoside II, c is agastachoside A, d is agastachoside B, and e is rhamnosyl icariside II).
The process flow diagram of the present invention is shown in figure 2.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages: (1) by adopting the process of the invention and adopting an absolute mass method for calculation, the purity of the epimedium low-glycoside component can be improved from 40 percent to more than 65 percent. (2) The invention indicates the dosage range of the cellulase in the enzymolysis step when different varieties of epimedium decoction pieces are fed, and the dosage of the cellulase can be effectively saved. (3) The method adds a chlorophyll removing step, solves the amplification effect of chlorophyll in industrial production, and ensures that an enzymolysis step and a column purification step are not influenced by the chlorophyll. (4) The invention adds a pretreatment step of the sample loading liquid and adopts a strategy of low-concentration ethanol sample loading, thereby solving the problem that the sample loading liquid is too turbid to block the resin column in industrial production, so that the production cannot be carried out.
Drawings
FIG. 1 is a flow chart of the prior art for preparing low-glycoside epimedium component;
FIG. 2 is a process flow diagram of the present invention.
Detailed Description
Example 1
(1) Taking 100g of Korean epimedium decoction pieces, adding 50% ethanol with 20 times volume, heating and refluxing for 2 times, each time for 2 hours, filtering and combining filtrates, recovering ethanol under reduced pressure, diluting the concentrated solution with pure water to the concentration of 0.2g decoction pieces/mL, and performing suction filtration to obtain supernatant and remove chlorophyll to obtain an enzymolysis stock solution;
(2) according to the cellulase: adding cellulase into the enzymolysis stock solution at the mass ratio of 1: 10, stirring at 65 + -3 deg.C for enzymolysis for 3h to obtain herba Epimedii total flavone enzymolysis solution;
(3) centrifuging the enzymolysis liquid at 4000rpm for 10min, discarding the supernatant, dissolving the precipitate with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging at 4000rpm for 10min again, discarding the precipitate, taking the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 65 deg.C for 12h to obtain crude product of icariin;
(4) redissolving the crude product of the epimedium low-glucoside by using 25 percent of ethanol in volume fraction until the concentration is 0.2g decoction pieces/mL, filtering and taking supernatant, redissolving the precipitate by using 25 percent of ethanol, filtering again, and merging the supernatant to obtain clear epimedium low-glucoside supernatant;
(5) NKA-9 type macroporous resin is adopted, and the ratio of the resin: purifying the decoction pieces with a weight ratio of 0.6: 1 by column purification, activating the resin with ethanol, loading the resin into the column by wet method, adsorbing with sample at 0.5BV/h, eluting with 1BV pure water, 6BV 40% ethanol, 3BV 60% ethanol, 5BV 80% ethanol at a flow rate of 1BV/h, collecting the 60% and 80% ethanol elution parts, respectively, recovering ethanol from the 60% ethanol elution part under reduced pressure, loading and eluting again according to the above method, and combining the two 80% ethanol elution parts; recovering ethanol under reduced pressure, and vacuum drying at 65 deg.C for 12 hr to obtain herba Epimedii low sugar glycoside component powder.
(6) The purity of the epimedium low-sugar-glycoside component powder is determined: weighing appropriate amount of herba Epimedii low-sugar glycoside powder, precisely weighing, adding methanol to desired volume, dissolving completely, detecting content of low-sugar glycoside component by HPLC, wherein the liquid phase is ThermoFisher U3000 high performance liquid chromatograph, the chromatographic column is Agilent ZORBAX SB-C18(250mm 4.6mm, 5 μm), the liquid phase is acetonitrile-water, wavelength is 270nm, column temperature is 30 deg.C, flow rate is 1.0mL/min, sample introduction amount is 10 μ L, and mobile phase gradient condition is as shown in Table below
Finally calculating by weight ratio to obtain epimedium low-glucoside powder with the purity of 71.5 percent.
Example 2
(1) Taking 100g of epimedium decoction pieces of the core leaf, adding 50% ethanol with the volume being 20 times that of the decoction pieces, heating and refluxing for 2 times, extracting for 2 hours each time, filtering and combining the filtrate, recovering the ethanol under reduced pressure, diluting the concentrated solution with pure water to the concentration of 0.2g decoction pieces/mL, and performing suction filtration to obtain supernatant fluid and remove chlorophyll to obtain enzymolysis stock solution;
(2) according to the cellulase: adding cellulase into the enzymolysis stock solution at the mass ratio of 1: 20, and stirring at 65 + -3 deg.C for enzymolysis for 3 hr to obtain herba Epimedii total flavone enzymolysis solution;
(3) centrifuging the enzymolysis liquid at 4000rpm for 10min, removing the supernatant, dissolving the precipitate with 95% ethanol to the concentration of 0.2g decoction pieces/mL, centrifuging at 4000rpm for 10min again, removing the precipitate, collecting the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 65 deg.C for 12h to obtain crude product of icariin;
(4) redissolving the crude product of the epimedium low-glycoside by using 25 percent of ethanol in volume fraction until the concentration is 0.2g decoction piece/mL, filtering to obtain supernatant, redissolving the precipitate by using 25 percent of ethanol, performing suction filtration again, and combining the supernatant to obtain clear epimedium low-glycoside supernatant;
(5) adopting ADS-17 type macroporous resin, and mixing the following components in percentage by weight: purifying decoction pieces by a column at a weight ratio of 0.6: 1, activating resin with ethanol, loading the resin into the column by a wet method, adsorbing by a sample at 0.5BV/h, eluting by 1BV pure water, 6BV 40% ethanol, 3BV 60% ethanol and 5BV 80% ethanol at a flow rate of 1BV/h, collecting 60% and 80% ethanol elution parts respectively, recovering ethanol from the 60% ethanol elution part under reduced pressure, eluting by the sample again according to the method, and combining the two 80% ethanol elution parts; recovering ethanol under reduced pressure, and vacuum drying at 65 deg.C for 12 hr to obtain herba Epimedii low sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 67.6% using the method in example 1.
Example 3
(1) Adding 50% ethanol 20 times the volume of 1kg of epimedium decoction pieces to the epimedium decoction pieces, heating and refluxing for 2 times, extracting for 2 hours each time, filtering and combining the filtrates, recovering ethanol under reduced pressure, diluting the concentrated solution with pure water to the concentration of 0.2g decoction pieces/mL, and performing suction filtration to obtain supernatant and remove chlorophyll to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of cellulase to epimedium decoction pieces of 1: 15, and stirring for enzymolysis at 65 +/-3 ℃ for 3 hours to obtain epimedium total flavone enzymolysis solution;
(3) centrifuging the enzymolysis liquid at 4000rpm for 10min, removing the supernatant, dissolving the precipitate with 95% ethanol to the concentration of 0.2g decoction pieces/mL, centrifuging at 4000rpm for 10min again, removing the precipitate, collecting the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 65 deg.C for 20h to obtain crude product of icariin;
(4) redissolving the crude product of the epimedium low-glycoside by using 25 percent of ethanol in volume fraction until the concentration is 0.2g decoction piece/mL, filtering to obtain supernatant, redissolving the precipitate by using 25 percent of ethanol, performing suction filtration again, and combining the supernatant to obtain clear epimedium low-glycoside supernatant;
(5) ADS-17 macroporous resin is adopted, and the resin: purifying decoction pieces by a column at a weight ratio of 0.8: 1, activating resin with ethanol, loading the resin into the column by a wet method, adsorbing by a sample at 0.5BV/h, eluting by 1BV pure water, 6BV 40% ethanol, 3BV 60% ethanol and 5BV 80% ethanol at a flow rate of 1BV/h, collecting 60% and 80% ethanol elution parts respectively, recovering ethanol from the 60% ethanol elution part under reduced pressure, eluting by the sample again according to the method, and combining the two 80% ethanol elution parts; recovering ethanol under reduced pressure, and vacuum drying at 75 deg.C for 20 hr to obtain herba Epimedii low sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 68.0% using the method in example 1.
Example 4
(1) Taking 8.5kg of epimedium brevicornum decoction pieces, adding 50% ethanol with the volume being 20 times that of the decoction pieces, heating and refluxing for 2 times, carrying out 2 hours each time, filtering and combining filtrates, recovering ethanol under reduced pressure, diluting a concentrated solution with pure water to the concentration of 0.4g decoction pieces/mL, sieving with a 120-mesh sieve, taking a supernatant, removing chlorophyll, and diluting a solution to 0.2g decoction pieces/mL to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of cellulase to epimedium decoction pieces of 1: 30, and stirring and performing enzymolysis for 3 hours at 65 +/-3 ℃ to obtain epimedium total flavone enzymolysis solution;
(3) centrifuging the enzymolysis solution by using a tubular centrifuge at the rotation speed of 16000rpm, discarding the supernatant, collecting the precipitate, dissolving the precipitate with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging again, discarding the precipitate, taking the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 75 ℃ for 24h to obtain a crude product of icariin;
(4) redissolving the crude product of the epimedium low-glycoside by using 25 percent of ethanol in volume fraction until the concentration is 0.2g decoction piece/mL, filtering to obtain supernatant, redissolving the precipitate by using 25 percent of ethanol, performing suction filtration again, and combining the supernatant to obtain clear epimedium low-glycoside supernatant;
(5) performing column purification by adopting NKA-9 type macroporous resin according to the weight ratio of resin to decoction pieces of 0.7: 1, performing column loading by a wet method after activating the resin by using ethanol, performing sample loading adsorption by 1BV/h, sequentially performing elution by using 2BV pure water, 7BV 40% ethanol, 4BV 60% ethanol and 6BV 80% ethanol at the flow rate of 2BV/h, respectively collecting 60% and 80% ethanol elution parts, performing sample loading elution again according to the method after recovering the ethanol from the 60% ethanol elution part under reduced pressure, and combining the two 80% ethanol elution parts; recovering ethanol under reduced pressure, and vacuum drying at 75 deg.C for 24 hr to obtain herba Epimedii low-sugar glycoside component powder.
(6) The purity of epimedium low-glucosidic powder measured by the method in example 1 was 65.3%.
Example 5
(1) Taking 10kg of epimedium herb decoction pieces, adding 50% ethanol with the volume being 20 times that of the decoction pieces, heating and refluxing for 2 times, carrying out 2 hours each time, filtering and combining filtrates, recovering ethanol under reduced pressure, diluting a concentrated solution with pure water to the concentration of 0.4g decoction pieces/mL, sieving with a 100-mesh sieve, taking a supernatant, removing chlorophyll, and diluting a solution to 0.2g decoction pieces/mL to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of 1: 40 of cellulase to herba Epimedii decoction pieces, and stirring at 65 + -3 deg.C for enzymolysis for 3h to obtain herba Epimedii total flavone enzymolysis solution;
(3) centrifuging the enzymolysis solution by using a tubular centrifuge at the rotation speed of 16000rpm, discarding the supernatant, collecting the precipitate, dissolving the precipitate with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging again, discarding the precipitate, taking the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 75 ℃ for 24h to obtain a crude product of icariin;
(4) redissolving the crude product of the epimedium low-glycoside by using 25 percent of ethanol in volume fraction until the concentration is 0.2g decoction piece/mL, filtering to obtain supernatant, redissolving the precipitate by using 25 percent of ethanol, performing suction filtration again, and combining the supernatant to obtain clear epimedium low-glycoside supernatant;
(5) performing column purification by using ADS-17 type macroporous resin according to a weight ratio of resin to decoction pieces of 0.8: 1, activating the resin with ethanol, filling the resin into a column by a wet method, adsorbing the resin by using a sample at 1BV/h, eluting the resin by using 2BV pure water, 7BV 40% ethanol, 4BV 60% ethanol and 6BV 80% ethanol at a flow rate of 2BV/h, respectively collecting elution parts of 60% and 80% ethanol, recovering ethanol from the elution part of 60% ethanol under reduced pressure, eluting the resin by using the sample again according to the method, and combining the elution parts of 80% ethanol twice; recovering ethanol under reduced pressure, and vacuum drying at 75 deg.C for 24 hr to obtain herba Epimedii low sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 68.4% using the method in example 1.
Example 6
(1) Taking 10kg of Korean epimedium decoction pieces, adding 50% ethanol with 20 times volume of the Korean epimedium decoction pieces, heating and refluxing for 2 times, each time for 2 hours, filtering and combining filtrates, recovering ethanol under reduced pressure, diluting a concentrated solution with pure water to the concentration of 0.4g decoction pieces/mL, sieving with a 50-mesh sieve to obtain a supernatant, removing chlorophyll, and diluting the solution to 0.2g decoction pieces/mL to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of 1: 20 of cellulase to herba Epimedii decoction pieces, and stirring at 65 + -3 deg.C for enzymolysis for 3h to obtain herba Epimedii total flavone enzymolysis solution;
(3) centrifuging the enzymolysis liquid by adopting a tubular centrifuge at the rotation speed of 16000rpm, discarding supernatant, collecting precipitate, dissolving with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging again, discarding precipitate, taking supernatant, recovering ethanol under reduced pressure, and vacuum drying at 75 ℃ for 24h to obtain a crude product of icariin;
(4) redissolving the crude product of the epimedium low-glucoside by using 25 percent of ethanol in volume fraction until the concentration is 0.2g decoction pieces/mL, filtering and taking supernatant, redissolving the precipitate by using 25 percent of ethanol, filtering again, and merging the supernatant to obtain clear epimedium low-glucoside supernatant;
(5) performing column purification by using ADS-7 type macroporous resin according to a weight ratio of resin to decoction pieces of 0.8: 1, activating the resin with ethanol, filling the resin into a column by a wet method, adsorbing the resin by using a sample at 1BV/h, eluting the resin by using 2BV pure water, 7BV 40% ethanol, 4BV 60% ethanol and 6BV 80% ethanol at a flow rate of 2BV/h, respectively collecting 60% and 80% ethanol elution parts, recovering ethanol from the 60% ethanol elution part under reduced pressure, eluting the resin by using the sample again according to the method, and combining the two 80% ethanol elution parts; recovering ethanol under reduced pressure, and vacuum drying at 75 deg.C for 24 hr to obtain herba Epimedii low sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 66.7% using the method in example 1.
Example 7
(1) Taking 15kg of epimedium herb decoction pieces, adding 50% ethanol with the volume being 20 times of that of the decoction pieces, heating and refluxing for 2 times, filtering and combining filtrates, recovering ethanol under reduced pressure, diluting a concentrated solution with pure water to the concentration of 0.5g decoction pieces/mL, centrifuging by adopting a tubular centrifuge, centrifuging at 16000rpm to obtain a supernatant, removing chlorophyll, and diluting the solution to 0.2g decoction pieces/mL to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of cellulase to epimedium decoction pieces of 1: 40, and stirring and performing enzymolysis for 3 hours at 65 +/-3 ℃ to obtain epimedium total flavone enzymolysis solution;
(3) centrifuging the enzymolysis solution by using a tubular centrifuge at the rotation speed of 16000rpm, discarding the supernatant, collecting the precipitate, dissolving the precipitate with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging again, discarding the precipitate, taking the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 75 ℃ for 24h to obtain a crude product of icariin;
(4) redissolving the crude product of the epimedium low-glucoside by using 28 percent of ethanol in volume fraction until the concentration is 0.2g decoction piece/mL, filtering and taking supernatant, redissolving the precipitate by using 28 percent of ethanol, filtering again, and merging the supernatant to obtain clear epimedium low-glucoside supernatant;
(5) ADS-7 type macroporous resin is adopted, column purification is carried out according to the weight ratio of resin to decoction pieces being 0.9: 1, the resin is activated by ethanol and then is packed into a column by a wet method, after sample loading adsorption at 2BV/h, elution is carried out sequentially by 2BV pure water, 6BV 40% ethanol, 3BV 60% ethanol and 5BV 80% ethanol at the flow rate of 2BV/h, elution parts of 60% ethanol and 80% ethanol are respectively collected, after the ethanol is recovered from the elution part of 60% ethanol under reduced pressure, sample loading elution is carried out again according to the method, and the elution parts of 80% ethanol at two times are combined; recovering ethanol under reduced pressure, and vacuum drying at 75 deg.C for 36 hr to obtain herba Epimedii low-sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 69.7% using the method in example 1.
Example 8
(1) Taking 100kg of epimedium herb decoction pieces, adding 50% ethanol with the volume being 20 times that of the decoction pieces, heating and refluxing for 2 times, extracting for 2 hours each time, filtering and combining filtrate, recovering ethanol under reduced pressure, diluting a concentrated solution to the concentration of 0.6g decoction pieces/mL by pure water, centrifuging by adopting a tubular centrifuge, centrifuging at 16000rpm to obtain a supernatant, removing chlorophyll, and diluting the solution to 0.2g decoction pieces/mL to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of 1: 50 of cellulase to herba Epimedii decoction pieces, and stirring at 65 + -3 deg.C for enzymolysis for 3h to obtain herba Epimedii total flavone enzymolysis solution;
(3) centrifuging the enzymolysis solution by using a tubular centrifuge at the rotation speed of 16000rpm, discarding the supernatant, collecting the precipitate, dissolving the precipitate with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging again, discarding the precipitate, taking the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 85 ℃ for 24h to obtain a crude product of icariin;
(4) redissolving the crude product of the epimedium low-glucoside by using 30 percent of ethanol in volume fraction until the concentration is 0.2g decoction pieces/mL, centrifuging by adopting a tubular centrifuge at 16000rpm to obtain supernatant, redissolving the precipitate by using 30 percent of ethanol, centrifuging again, and combining the supernatant to obtain clear epimedium low-glucoside supernatant;
(5) performing column purification by using ADS-7 type macroporous resin according to the weight ratio of resin to decoction pieces of 1: 1, activating the resin with ethanol, filling the resin into a column by using a wet method, adsorbing the resin by using a sample at 2BV/h, eluting the resin by using 2BV pure water, 6BV 40% ethanol, 3BV 60% ethanol and 5BV 80% ethanol at the flow rate of 2BV/h in sequence, collecting the elution parts of 60% and 80% ethanol respectively, recovering the ethanol from the elution part of 60% ethanol under reduced pressure, eluting the resin by using the sample again according to the method, and combining the elution parts of 80% ethanol twice; recovering ethanol under reduced pressure, and vacuum drying at 85 deg.C for 48 hr to obtain herba Epimedii low-sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 69.4% using the method in example 1.
Example 9
(1) Taking 300kg of epimedium herb decoction pieces, adding 50% ethanol with the volume being 20 times that of the decoction pieces, heating and refluxing for 2 times, extracting for 2 hours each time, filtering and combining filtrate, recovering ethanol under reduced pressure, diluting a concentrated solution to the concentration of 0.6g decoction piece/mL by pure water, centrifuging by adopting a tubular centrifuge, centrifuging at 16000rpm to obtain a supernatant, removing chlorophyll, and diluting the solution to 0.2g decoction piece/mL to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of cellulase to epimedium decoction pieces of 1: 50, and stirring and performing enzymolysis for 3 hours at 65 +/-3 ℃ to obtain epimedium total flavone enzymolysis solution;
(3) centrifuging the enzymolysis solution by using a tubular centrifuge at the rotation speed of 16000rpm, discarding the supernatant, collecting the precipitate, dissolving the precipitate with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging again, discarding the precipitate, taking the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 85 ℃ for 24h to obtain a crude product of icariin;
(4) redissolving the crude product of the epimedium low-sugar glucoside by using 30 percent of ethanol in volume fraction until the concentration is 0.2g decoction pieces/mL, centrifuging by adopting a tubular centrifuge at 16000rpm to obtain supernatant, redissolving the precipitate by using 30 percent of ethanol, centrifuging again, and combining the supernatant to obtain clear epimedium low-sugar glucoside supernatant;
(5) performing column purification by using ADS-17 type macroporous resin according to a weight ratio of resin to decoction pieces of 1: 1, activating the resin with ethanol, filling the resin into a column by using a wet method, adsorbing the resin by using a sample at 2BV/h, eluting the resin by using 2BV pure water, 6BV 40% ethanol, 3BV 60% ethanol and 5BV 80% ethanol at a flow rate of 2BV/h in sequence, collecting elution parts of 60% and 80% ethanol respectively, recovering ethanol from the elution parts of 60% ethanol under reduced pressure, eluting the resin by using the sample again according to the method, and combining the elution parts of 80% ethanol twice; recovering ethanol under reduced pressure, and vacuum drying at 85 deg.C for 48 hr to obtain herba Epimedii low sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 69.0% using the method in example 1.
Example 10
(1) Taking 1000kg of epimedium herb decoction pieces, adding 50% ethanol with the volume being 20 times that of the decoction pieces, heating and refluxing for 2 times, extracting for 2 hours each time, filtering and combining filtrate, recovering ethanol under reduced pressure, diluting a concentrated solution with pure water to the concentration of 0.8g decoction pieces/mL, centrifuging by adopting a tubular centrifuge, centrifuging at 16000rpm to obtain a supernatant, removing chlorophyll, and diluting the solution to 0.2g decoction pieces/mL to obtain an enzymolysis stock solution;
(2) adding cellulase into the enzymolysis stock solution according to the mass ratio of 1: 50 of cellulase to herba Epimedii decoction pieces, and stirring at 65 + -3 deg.C for enzymolysis for 3h to obtain herba Epimedii total flavone enzymolysis solution;
(3) centrifuging the enzymolysis solution by using a tubular centrifuge at the rotation speed of 16000rpm, discarding the supernatant, collecting the precipitate, dissolving the precipitate with 95% ethanol until the concentration is 0.2g decoction pieces/mL, centrifuging again, discarding the precipitate, taking the supernatant, recovering ethanol under reduced pressure, and vacuum drying at 85 ℃ for 24h to obtain a crude product of icariin;
(4) redissolving the crude product of the epimedium low-sugar glucoside by using 30 percent of ethanol in volume fraction until the concentration is 0.2g decoction pieces/mL, centrifuging by adopting a tubular centrifuge at 16000rpm to obtain supernatant, redissolving the precipitate by using 30 percent of ethanol, centrifuging again, and combining the supernatant to obtain clear epimedium low-sugar glucoside supernatant;
(5) ADS-17 type macroporous resin is adopted, column purification is carried out according to the weight ratio of resin to decoction pieces being 1: 1, the resin is activated by ethanol and then is packed into a column by a wet method, after sample loading adsorption at 2BV/h, elution is carried out sequentially by 2BV pure water, 6BV 40% ethanol, 3BV 60% ethanol and 5BV 80% ethanol at the flow rate of 2BV/h, elution parts of 60% ethanol and 80% ethanol are respectively collected, after the ethanol is recovered from the elution part of 60% ethanol under reduced pressure, sample loading elution is carried out again according to the method, and the elution parts of 80% ethanol at twice are combined; recovering ethanol under reduced pressure, and vacuum drying at 85 deg.C for 48 hr to obtain herba Epimedii low sugar glycoside component powder.
(6) The purity of icariin powder was determined to be 68.1% using the method in example 1.
Claims (10)
1. A method for preparing a high-purity epimedium flavone low-glycoside component is characterized by comprising the following steps of
(1) Heating and refluxing with ethanol solution to extract herba Epimedii decoction pieces, and recovering ethanol under reduced pressure to obtain concentrated solution;
(2) dissolving the concentrated solution with water for dilution, and removing chlorophyll in the solution by a physical method to obtain an enzymolysis stock solution;
(3) adding cellulase into the enzymolysis stock solution, stirring for enzymolysis, and completely reacting to obtain an enzymolysis solution;
(4) centrifuging the enzymolysis solution, removing supernatant, extracting herba Epimedii low-glycoside component in the precipitate with ethanol solution, centrifuging again to obtain supernatant, recovering ethanol under reduced pressure, and drying to obtain herba Epimedii low-glycoside component crude product;
(5) re-dissolving the coarse epimedium low-sugar component with low-concentration ethanol, fully dissolving, and then carrying out sample loading pretreatment to obtain clear epimedium low-sugar component sample loading liquid;
(6) adsorbing the sample solution with macroporous resin, sequentially eluting with water, 35-45% ethanol, 55-65% ethanol, and 75-85% ethanol for the first time, collecting 55-65% and 75-85% ethanol eluate, recovering ethanol from 55-65% ethanol eluate under reduced pressure, diluting with pure water, adsorbing with macroporous resin, and eluting again by the same method;
(7) mixing the two 75-85% ethanol eluates, recovering ethanol under reduced pressure, and drying to obtain high-purity epimedium low-sugar glycoside component.
2. The method of claim 1, wherein the ethanol solution of step (1) is a 40-70% ethanol solution.
3. The method as claimed in claim 1, wherein the epimedium decoction pieces used in step (1) are selected from epimedium koreanum, epimedium cardioides and epimedium dauricum.
4. The method of claim 1, wherein the concentration of the rotary solution used for centrifugation in step (2) is 0.2-0.8 g decoction pieces/mL, and the physical method for chlorophyll removal comprises centrifugation, suction filtration, and sieving; if centrifugation is selected, the rotating speed is 3500-16000 rpm; if a screen is selected, the aperture of the screen is 50-120 meshes.
5. The method as claimed in claim 1, wherein the mass ratio of the cellulase to the epimedium decoction pieces in the step (3) is 1: 10 to 1: 50, the mass ratio of the cellulase to the epimedium decoction pieces is 1: 10 to 1: 20 if the epimedium koreanum is used, and the mass ratio of the cellulase to the epimedium decoction pieces is 1: 20 to 1: 50 if the epimedium brevicornum or the epimedium dauricum is used.
6. The method of claim 1, wherein the ethanol solution of step (4) is an 80-99.5% ethanol solution.
7. The method of claim 1, wherein the volume fraction of ethanol in the low-concentration ethanol in the step (5) is 25-30%, and the pre-treatment method for sample loading comprises centrifugation and suction filtration.
8. The method according to claim 1, wherein the centrifugal rotation speed during the pre-sample treatment in the step (5) is 3500-16000 rpm.
9. The method of claim 1, wherein the macroporous resin used in step (6) is NKA-9, ADS-17 or ADS-7 type macroporous adsorbent resin, and the amount of macroporous resin is: the weight ratio of the decoction pieces is 0.6: 1-1: 1.
10. The method according to claim 1, wherein in the step (7), the drying condition is vacuum drying, the temperature is controlled to be 65-85 ℃, and the drying time is 12-48 h.
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