CN104496783B - A kind of isolation and purification method of 4 '-O-methyl Bavachalcone B monomer - Google Patents
A kind of isolation and purification method of 4 '-O-methyl Bavachalcone B monomer Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/80—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/81—Separation; Purification; Stabilisation; Use of additives by change in the physical state, e.g. crystallisation
Abstract
The present invention relates to the isolation and purification method of a kind of 4'O methyl Bavachalcone B monomer, belong to the separating and purifying technology field of active ingredient of Chinese herbs.The present invention is with the dry mature fruit of legumes psoraleae as raw material, and by ethanol solution extraction, ethyl acetate extraction, silica gel column chromatography, polyamide column chromatography, methanol dissolution filter, high performance preparative liquid chromatography separates, Product recycling obtains 4'O methyl Bavachalcone B monomer product.The present invention, by optimum technique and Parameter Conditions, makes the content of 4'O methyl Bavachalcone B in product reach more than 98%；Whole stable technical process, easy and simple to handle, separation efficiency is high, with low cost, can realize a large amount of high-purity and separate preparation 4'O methyl Bavachalcone B monomer.
Description
Technical field
The present invention relates to the preparation method of a kind of plant compound monomer, a kind of isolation and purification method extracting 4'-O-methyl Bavachalcone B monomer from Fructus Psoraleae, belong to the separating and purifying technology field of active ingredient of Chinese herbs.
Background technology
Fructus Psoraleae (formal name used at school: Psoralea
Corylifolia Linn.) have another name called: Fructus Psoraleae, the solid fat of mother-in-law, Semen Allii Tuberosi recklessly.Belong to Rosales, the annual upright draft of pulse family, high 60150cm.Leaf is single leaf, long 7-8mm；The long 2-4.5cm of petiole, leaf width egg shape, inflorescence axil is raw, has colored 10-30, corolla yellow or blueness, and petal substantially has lobe handle, vexil obovate, and pod is avette, flower, really the phase 7-10 month.Fructus Psoraleae is the dry fruit of legumes psoraleae, mainly containing chemical compositions such as Coumarins, flavonoid, monoterpene phenols, wherein, 4'-O-methyl Bavachalcone B is the flavonoid active component in corylifolia L, there is treatment dermatosis, antitumor, effect of insecticidal and antibacterial.4'-O-methyl Bavachalcone B be again 4'-O-Methylbroussochalcone
B, molecular formula is C21H22O4, molecular weight is 338.4, and structural formula is as follows:
Through retrieval, the pertinent literature currently for Constituents in Fruits of Psoralea corylifolia L research mainly has:
1) " Fructus Psoraleae active site ethyl acetate extract chemical constitution study " (in December, 2011, " Chongqing Chinese medicine research ", 2nd phase (total 64th phase) 13), disclose employing the method such as the most anti-phase silica gel column chromatography and preparative hplc separate, use VU, IR, HPLC-MS,1HNMR、13The spectral methods such as CNMR carry out Structural Identification, it is divided into obtain 9 compounds, it is respectively isopsoralen, psoralen, psoralidine, Bavachalcone, bavachinin, Corylifolinin, isobavachin, Coryfolin, corylin；I.e. Fructus Psoraleae active site ethyl acetate extract is mainly composed of legumin class and flavones ingredient.
null2) " research of Constituents in Fruits of Psoralea corylifolia L " (the 4th phase of volume 25，In July, 2005，Yunnan Normal University)，Disclosing extraction separation method is: be dried Fructus Psoraleae seed 1kg,Extract four times with industrial alcohol merceration，Leachate concentrating under reduced pressure obtains ethanol extraction，Dissolve with methanol (9:1)，Then extract successively with petroleum ether and chloroform，After chloroform portion recycling design，With 80-100 mesh silica gel mixed sample，Carry out silica gel column chromatography，Respectively with petroleum ether: acetone (100:0-0:100)，Chloroform: methanol (100:0-0:100) gradient elution，Thin layer chromatography detects，Merge into 20 parts，4th part obtains 4 kinds of amorphous powders through silica gel column chromatography purification respectively with preparing thin layer chromatography，It is respectively Bavachalcone through Structural Identification、Bavachromene、Pinitol and hydroxy benzaldehyde.
As can be seen here, in the existing method for Constituents in Fruits of Psoralea corylifolia L research, all it is not directed to the extraction to chemical composition single in Fructus Psoraleae and separates, the most more have no the relevant report of industrial separation purification 4'-O-methyl Bavachalcone B monomer from Fructus Psoraleae.
Summary of the invention
It is an object of the invention to provide the method that the sharp separation of a kind of simplicity prepares high-load high-purity 4'-O-methyl Bavachalcone B.The method separation efficiency is high, with short production cycle, and process stabilizing is easy and simple to handle, with low cost, and the high-purity (area normalization method more than 98%) that can realize a large amount of 4'-O-methyl Bavachalcone B monomer separates preparation.
In order to realize foregoing invention purpose, the technical solution used in the present invention is as follows:
A kind of isolation and purification method of 4'-O-methyl Bavachalcone B monomer, it is characterized in that with the dry mature fruit of legumes psoraleae as raw material, by ethanol solution extraction, ethyl acetate extraction, silica gel column chromatography, polyamide column chromatography, methanol dissolution filter, high performance preparative liquid chromatography separates, Product recycling obtains 4'-O-methyl Bavachalcone B monomer product, specifically includes following processing step:
1) extract
Corylifolia L is ground into the coarse powder of 1～4mm, is incorporated as quality of medicinal material 8～the ethanol solution of 10 times of volumes, extract 3～4 times, each 2～3 hours, reclaim ethanol, merge filtrate；
The concentration of volume percent of described ethanol solution is 90～95%；
2) extraction
By step 1) filtrate of gained, add isopyknic ethyl acetate and extract 3～4 times, point take upper solution, be concentrated to dryness, obtain concentrated solution；
3) silica gel column chromatography
By step 2) concentrated solution of gained adds the silica gel mixed sample of 8 times of weight, and 45 DEG C are dried and mix sample silica gel, with petroleum ether: ethyl acetate volume ratio 5:1 carries out eluting, and column chromatography eluent 45 DEG C is concentrated to dryness, and obtains extractum；This step can remove a large amount of impurity and to reach trace product enriched；
4) polyamide column chromatography
According to extractum quality: solvent volume=1g:5～10ml, the extractum of step 3) gained being added acetic acid ethyl dissolution, the solution of gained adds the polyamide of 4 times of volumes and mixes sample, 45 DEG C be dried mix sample polyamide, carrying out eluting with dichloromethane, column chromatography eluent 40 DEG C is concentrated to dryness, and obtains solids；This step can remove other non-flavone components；
5) dissolution filter
According to solids quality: solvent volume=1g:20～40ml, step 4) gained solids adding methanol and dissolves, filter lysate, filtrate is i.e. the raw material preparing 4'-O-methyl Bavachalcone B monomer；
6) prepared by HPLC
Chromatographic column filler is: C18Filler；
Flowing is mutually: methanol-water solution V/V=83:17；
Take the filtrate sample introduction of step 5) gained, sample size is calculated as 2.0-3.0g/ pin with the dry product weight contained by filtrate, carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, detection wavelength is 245nm, collects target compound and prepares solution；
7) Product recycling
The HPLC of step 6) gained being prepared solution, reclaims methanol, reddish-orange crystals separates out from remaining aqueous solution, lets cool to filter and i.e. obtains 4'-O-methyl Bavachalcone B monomer.
It is an advantage of the current invention that:
1. the present invention makes 4'-O-methyl Bavachalcone B be transferred in ethyl acetate solution from extraction concentrated aqueous solution by extraction, not only increases target concentration, simultaneously because ethyl acetate is prone to concentrate, also reduces energy consumption.
2. the present invention uses preparative high performance liquid chromatography system to carry out isolated and purified to 4'-O-methyl Bavachalcone B monomer, reach good separating effect, and by online ultraviolet monitoring, collect 4'-O-methyl Bavachalcone B monomer targetedly, with clearly defined objective, simultaneously facilitating control product quality, product purity is up to more than 98%.
3. due to high performance liquid chromatography, the color and luster of sample, purity etc. are required higher, the extracting solution obtained by simple process can not direct injected, if therefore before carrying out high performance liquid chromatography separation, according to the inventive method step, sample introduction solution is not carried out pre-treatment, the most not only separating effect is bad, but also instrument may be caused and have a strong impact on (as shortened the life-span etc.), and then increase the production cost of product.
4. for the physicochemical property of various compositions present in corylifolia L, the inventive method pass through abovementioned steps 1), 2), 3), 4) order arrange in pairs or groups, and suitable parameter combination, the compositions such as 4'-O-methyl Bavachalcone B are effectively extracted, and eliminate a large amount of impurity, it is derived from entering the sample solution of preparative high performance liquid chromatography system, unlikely highly effective liquid phase chromatographic system is caused the biggest impact, extend its use cycle as far as possible, save production cost.
5. in high performance liquid chromatography separation process, selection of each chromatographic condition and combinations thereof is critically important, because it directly affects the appearance time of material, peak shape etc.；Chromatographic condition mainly includes chromatographic column (including filler, column length, column temperature etc.), flowing phase (including composition, flow velocity etc.), detector and detection wavelength etc..The present invention is by substantial amounts of experimental study and relative analysis, it is determined that above-described chromatographic condition, so that the appearance time of material, peak shape, separating effect etc. reach optimization, it is achieved that the high efficiency separation of 4'-O-methyl Bavachalcone B monomer.
6. the present invention is by optimum technique and Parameter Conditions, makes the content of 4'-O-methyl Bavachalcone B in product reach more than 98%；Whole stable technical process, easy and simple to handle, separation efficiency is high, with low cost, can realize a large amount of high-purity and separate preparation 4'-O-methyl Bavachalcone B monomer.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates of the embodiment of the present invention 1 gained 4'-O-methyl Bavachalcone B monomer product.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in further detail, but should not be interpreted as this present invention is limited only to following embodiment.
In following each embodiment, the purity of finished product 4'-O-methyl Bavachalcone B monomer rechecks inverse analysis type liquid chromatograph (RP-HPLC) method that all uses, and chromatographic condition is as follows:
Filler is C18, flowing is methanol-water solution (87:13) mutually, and column temperature is 30 DEG C, and flow velocity is 1.0ml/min, and detection wavelength is 245 nm.
Embodiment
1
The isolation and purification method of a kind of 4'-O-methyl Bavachalcone B monomer, comprises the technical steps that:
1) extract
Corylifolia L 45kg is ground into the coarse powder of 1～4mm, and adding 360L concentration of volume percent is the ethanol solution of 90%, extracts 3 times, each 2 hours, reclaims ethanol, merge filtrate, altogether 20000ml；
2) extraction
By step 1) filtrate of gained, add isopyknic ethyl acetate and extract 3 times, point take upper solution, be concentrated to dryness, obtain concentrated solution 5000ml；
3) silica gel column chromatography
By step 2) concentrated solution of gained adds the silica gel mixed sample of 8 times of weight, and 45 DEG C are dried and mix sample silica gel, and with petroleum ether: ethyl acetate volume ratio (5:1) carries out eluting, column chromatography eluent 45 DEG C is concentrated to dryness, and obtains extractum 100 grams；
4) polyamide column chromatography
The extractum of step 3) gained adds 500 ml acetic acid ethyl dissolutions, and the solution of gained adds the polyamide of 4 times of volumes and mixes sample, and 45 DEG C are dried and mix sample polyamide, carry out eluting with dichloromethane, and column chromatography eluent 40 DEG C is concentrated to dryness, and obtains solids 20 grams；
5) dissolution filter
Step 4) gained solids adds 500ml methanol and dissolves, and filters lysate, obtains filtrate 490ml；
6) prepared by HPLC
Employing filler is C18Chromatographic column, post specification is 50cm × 10cm；
Flowing is mutually: methanol-water solution V/V(87:13)；
Detection wavelength is 245nm, ambient operation；
Taking the filtrate sample introduction of step 5) gained, sample size is 2g/ pin (in terms of the dry product weight contained by filtrate), carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, and collection obtains target compound and prepares solution 6000ml；
Before carrying out high performance preparative liquid chromatography separation, the peak shape of 4'-O-methyl Bavachalcone B monomer in high performance liquid chromatography is determined by liquid-mass chromatography method (chromatographic condition is ibid), take step 5) gained filtrate sample introduction, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to Mass Spectrometer Method result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatograph.
7) Product recycling
The HPLC of step 6) gained prepares solution, reclaims methanol, and reddish-orange crystals separates out from remaining aqueous solution, lets cool to filter and i.e. obtains 4'-O-methyl Bavachalcone B monomer product 8.0 grams.
About 15 days whole production procedure used times.
Calculating product yield is (8.0/45000) × 100%=0.018%.
By changing mobile phase composition, utilizing inverse analysis type liquid chromatograph (RP-HPLC) to recheck product purity, recording result is 99.90%.
Embodiment
2
The isolation and purification method of a kind of 4'-O-methyl Bavachalcone B monomer, comprises the technical steps that:
1) extract
Corylifolia L 60kg is ground into the coarse powder of 1～4mm, and adding 500L concentration of volume percent is the ethanol solution of 95%, extracts 3 times, each 2 hours, reclaims ethanol, merge filtrate, altogether 15000ml；
2) extraction
By step 1) filtrate of gained, add isopyknic ethyl acetate and extract 3 times, point take upper solution, be concentrated to dryness, obtain concentrated solution 6000ml；
3) silica gel column chromatography
By step 2) concentrated solution of gained adds the silica gel mixed sample of 8 times of weight, and 45 DEG C are dried and mix sample silica gel, and with petroleum ether: ethyl acetate volume ratio (5:1) carries out eluting, column chromatography eluent 45 DEG C is concentrated to dryness, and obtains extractum 150 grams；
4) polyamide column chromatography
The extractum of step 3) gained is added 1200ml acetic acid ethyl dissolution, and the solution of gained adds the polyamide of 4 times of volumes and mixes sample, and 45 DEG C are dried and mix sample polyamide, carry out eluting with dichloromethane, and column chromatography eluent 40 DEG C is concentrated to dryness, and obtains solids 30 grams；
5) dissolution filter
Step 4) gained solids adds 600ml methanol and dissolves, and filters lysate, obtains filtrate 590ml；
6) prepared by HPLC
Employing filler is C18Chromatographic column, post specification is 50cm × 10cm；
Flowing is mutually: methanol-water solution V/V=(87:13)；
Detection wavelength is 245nm, ambient operation；
Taking the filtrate sample introduction of step 5) gained, sample size is 2.5g/ pin (in terms of the dry product weight contained by filtrate), carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, and collection obtains target compound and prepares solution 8000ml；
Before carrying out high performance preparative liquid chromatography separation, the peak shape of 4'-O-methyl Bavachalcone B monomer in high performance liquid chromatography is determined by liquid-mass chromatography method (chromatographic condition is ibid), take the filtrate sample introduction of step 5) gained, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to Mass Spectrometer Method result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatograph.
7) Product recycling
The HPLC of step 6) gained prepares solution, reclaims methanol, and remaining aqueous solution has substantial amounts of reddish-orange crystals to separate out, and lets cool to filter and i.e. obtains 4'-O-methyl Bavachalcone B monomer product 12.0 grams.
About 12 days whole production procedure used times.
Calculating product yield is (12.0/60000) × 100%=0.02%.
By changing mobile phase composition, utilizing inverse analysis type liquid chromatograph (RP-HPLC) to recheck product purity, recording result is 99.10%.
Embodiment
3
The isolation and purification method of a kind of 4'-O-methyl Bavachalcone B monomer, comprises the technical steps that:
1) extract
Corylifolia L 80kg is ground into the coarse powder of 1～4mm, and adding 800L concentration of volume percent is the ethanol solution of 95%, extracts 4 times, each 2 hours, reclaims ethanol, merge filtrate, altogether 20000ml；
2) extraction
By step 1) filtrate of gained, add isopyknic ethyl acetate and extract 3 times, point take upper solution, be concentrated to dryness, obtain concentrated solution 9000ml；
3) silica gel column chromatography
By step 2) concentrated solution of gained adds the silica gel mixed sample of 8 times of weight, and 45 DEG C are dried and mix sample silica gel, and with petroleum ether: ethyl acetate volume ratio (5:1) carries out eluting, column chromatography eluent 45 DEG C is concentrated to dryness, and obtains extractum 250 grams；
4) polyamide column chromatography
The extractum of step 3) gained is added 2500ml acetic acid ethyl dissolution, and the solution of gained adds the polyamide of 4 times of volumes and mixes sample, and 45 DEG C are dried and mix sample polyamide, carry out eluting with dichloromethane, and column chromatography eluent 40 DEG C is concentrated to dryness, and obtains solids 45 grams；
5) dissolution filter
Step 4) gained solids adds 1800ml methanol and dissolves, and filters lysate, obtains filtrate 1780ml；
6) prepared by HPLC
Employing filler is C18Chromatographic column, post specification is 100cm × 20cm；
Flowing phase composition is: methanol-water solution V/V(87:13)
Detection wavelength is 245nm, ambient operation；
Taking the filtrate sample introduction of step 5) gained, sample size is 5.0g/ pin (in terms of the dry product weight contained by filtrate), carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, and collection obtains target compound and prepares solution 25000ml；
Before carrying out high performance preparative liquid chromatography separation, the peak shape of 4'-O-methyl Bavachalcone B monomer in high performance liquid chromatography is determined by liquid-mass chromatography method (chromatographic condition is ibid), take the filtrate sample introduction of step 5) gained, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to Mass Spectrometer Method result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatograph.
7) Product recycling
The HPLC of step 6) gained prepares solution, reclaims methanol, and reddish-orange crystals separates out from remaining aqueous solution, lets cool to filter and i.e. obtains 4'-O-methyl Bavachalcone B monomer product 18.0 grams.
About 12 days whole production procedure used times.
Calculating product yield is (18.0/80000) × 100%=0.0225%.
By changing mobile phase composition, utilizing inverse analysis type liquid chromatograph (RP-HPLC) to recheck product purity, recording result is 99.50%.
Claims (5)
1. the isolation and purification method of a 4'-O-methyl Bavachalcone B monomer, it is characterized in that with the dry mature fruit of legumes psoraleae as raw material, by ethanol solution extraction, ethyl acetate extraction, silica gel column chromatography, polyamide column chromatography, methanol dissolution filter, high performance preparative liquid chromatography separates, Product recycling obtains 4'-O-methyl Bavachalcone B monomer product, specifically includes following processing step:
1) extract
Corylifolia L is ground into the coarse powder of 1～4mm, is incorporated as quality of medicinal material 8～the ethanol solution of 10 times of volumes, extract 3～4 times, each 2～3 hours, reclaim ethanol, merge filtrate；
The concentration of volume percent of described ethanol solution is 90～95%；
2) extraction
By step 1) filtrate of gained, add isopyknic ethyl acetate and extract 3～4 times, point take upper solution, be concentrated to dryness, obtain concentrated solution；
3) silica gel column chromatography
By step 2) concentrated solution of gained adds the silica gel mixed sample of 8 times of weight, and 45 DEG C are dried and mix sample silica gel, with petroleum ether: ethyl acetate volume ratio 5:1 carries out eluting, and column chromatography eluent 45 DEG C is concentrated to dryness, and obtains extractum；
4) polyamide column chromatography
According to extractum quality: solvent volume=1g:5～10ml, the extractum of step 3) gained being added acetic acid ethyl dissolution, the solution of gained adds the polyamide of 4 times of volumes and mixes sample, 45 DEG C be dried mix sample polyamide, carrying out eluting with dichloromethane, column chromatography eluent 40 DEG C is concentrated to dryness, and obtains solids；
5) dissolution filter
According to solids quality: solvent volume=1g:20～40ml, step 4) gained solids adding methanol and dissolves, filter lysate, filtrate is i.e. the raw material preparing 4'-O-methyl Bavachalcone B monomer；
6) prepared by HPLC
Chromatographic column filler is: C18Filler；
Flowing is mutually: methanol-water solution V/V=87:13；
Take the filtrate sample introduction of step 5) gained, sample size is calculated as 2.0-3.0g/ pin with the dry product weight contained by filtrate, carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, detection wavelength is 245nm, collects target compound and prepares solution；
7) Product recycling
The HPLC of step 6) gained being prepared solution, reclaims methanol, reddish-orange crystals separates out from remaining aqueous solution, lets cool to filter and i.e. obtains 4'-O-methyl Bavachalcone B monomer.
The isolation and purification method of a kind of 4'-O-methyl Bavachalcone B monomer the most according to claim 1, it is characterised in that comprise the technical steps that:
1) extract
Corylifolia L 45kg is ground into the coarse powder of 1～4mm, and adding 360L concentration of volume percent is the ethanol solution of 90%, extracts 3 times, each 2 hours, reclaims ethanol, merge filtrate, altogether 20000ml；
2) extraction
By step 1) filtrate of gained, add isopyknic ethyl acetate and extract 3 times, point take upper solution, be concentrated to dryness, obtain concentrated solution 5000ml；
3) silica gel column chromatography
By step 2) concentrated solution of gained adds the silica gel mixed sample of 8 times of weight, and 45 DEG C are dried and mix sample silica gel, and with petroleum ether: ethyl acetate volume ratio 5:1 carries out eluting, column chromatography eluent 45 DEG C is concentrated to dryness, and obtains extractum 100 grams；
4) polyamide column chromatography
The extractum of step 3) gained is added 500
Ml acetic acid ethyl dissolution, the solution of gained adds the polyamide of 4 times of volumes and mixes sample, and 45 DEG C are dried and mix sample polyamide, carry out eluting with dichloromethane, and column chromatography eluent 40 DEG C is concentrated to dryness, and obtains solids 20 grams；
5) dissolution filter
Step 4) gained solids adds 500ml methanol and dissolves, and filters lysate, obtains filtrate 490ml；
6) prepared by HPLC
Employing filler is C18Chromatographic column, post specification is 50cm × 10cm；
Flowing is mutually: methanol-water solution V/V=87:13；
Detection wavelength is 245nm, ambient operation；
Taking the filtrate sample introduction of step 5) gained, sample size is calculated as 2g/ pin with the dry product weight contained by filtrate, carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, and collection obtains target compound and prepares solution 6000ml；
7) Product recycling
The HPLC of step 6) gained prepares solution, reclaims methanol, and reddish-orange crystals separates out from remaining aqueous solution, lets cool to filter and i.e. obtains 4'-O-methyl Bavachalcone B monomer product 8.0 grams；
About 15 days whole production procedure used times；
Calculating product yield is 8.0/45000 × 100%=0.018%；
By changing mobile phase composition, utilizing inverse analysis type liquid chromatograph RP-HPLC to recheck product purity, recording result is 99.90%.
The isolation and purification method of a kind of 4'-O-methyl Bavachalcone B monomer the most according to claim 1, it is characterised in that comprise the technical steps that:
1) extract
Corylifolia L 60kg is ground into the coarse powder of 1～4mm, and adding 500L concentration of volume percent is the ethanol solution of 95%, extracts 3 times, each 2 hours, reclaims ethanol, merge filtrate, altogether 15000ml；
2) extraction
By step 1) filtrate of gained, add isopyknic ethyl acetate and extract 3 times, point take upper solution, be concentrated to dryness, obtain concentrated solution 6000ml；
3) silica gel column chromatography
By step 2) concentrated solution of gained adds the silica gel mixed sample of 8 times of weight, and 45 DEG C are dried and mix sample silica gel, and with petroleum ether: ethyl acetate volume ratio 5:1 carries out eluting, column chromatography eluent 45 DEG C is concentrated to dryness, and obtains extractum 150 grams；
4) polyamide column chromatography
The extractum of step 3) gained is added 1200ml acetic acid ethyl dissolution, and the solution of gained adds the polyamide of 4 times of volumes and mixes sample, and 45 DEG C are dried and mix sample polyamide, carry out eluting with dichloromethane, and column chromatography eluent 40 DEG C is concentrated to dryness, and obtains solids 30 grams；
5) dissolution filter
Step 4) gained solids adds 600ml methanol and dissolves, and filters lysate, obtains filtrate 590ml；
6) prepared by HPLC
Employing filler is C18Chromatographic column, post specification is 50cm × 10cm；
Flowing is mutually: methanol-water solution V/V=87:13；
Detection wavelength is 245nm, ambient operation；
Taking the filtrate sample introduction of step 5) gained, sample size is calculated as 2.5g/ pin with the dry product weight contained by filtrate, carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, and collection obtains target compound and prepares solution 8000ml；
7) Product recycling
The HPLC of step 6) gained prepares solution, reclaims methanol, and remaining aqueous solution has substantial amounts of reddish-orange crystals to separate out, and lets cool to filter and i.e. obtains 4'-O-methyl Bavachalcone B monomer product 12.0 grams；
About 12 days whole production procedure used times；
Calculating product yield is 12.0/60000 × 100%=0.02%；
By changing mobile phase composition, utilizing inverse analysis type liquid chromatograph RP-HPLC to recheck product purity, recording result is 99.10%.
4. according to the isolation and purification method of a kind of 4'-O-methyl Bavachalcone B monomer described in Claims 2 or 3, it is characterised in that: the chromatographic condition that described inverse analysis type liquid chromatograph RP-HPLC carries out purity reinspection is as follows:
Filler is C18, flowing is methanol-water solution V/V=87:13 mutually, and column temperature is 30 DEG C, and flow velocity is 1.0ml/min, and detection wavelength is 245nm.
5. according to the isolation and purification method of a kind of 4'-O-methyl Bavachalcone B monomer described in Claims 2 or 3, it is characterized in that: before carrying out high performance preparative liquid chromatography separation, the peak shape of 4'-O-methyl Bavachalcone B monomer in high performance liquid chromatography is determined by liquid-mass chromatography method, i.e. take step 5) gained filtering solution sample introduction, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to Mass Spectrometer Method result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatograph.
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