CN104496783A - Method for separating and purifying 4'-O-methyl bavachalcone B monomers - Google Patents
Method for separating and purifying 4'-O-methyl bavachalcone B monomers Download PDFInfo
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- CN104496783A CN104496783A CN201410839484.9A CN201410839484A CN104496783A CN 104496783 A CN104496783 A CN 104496783A CN 201410839484 A CN201410839484 A CN 201410839484A CN 104496783 A CN104496783 A CN 104496783A
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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 invention relates to a method for separating and purifying 4'-O-methyl bavachalcone B monomers, which belongs to the technical field of separation and purification of active ingredients of traditional Chinese medicines. The method disclosed by the invention is implemented by taking dried ripe fruits of a leguminous plant fructus psoraleae through the steps of ethanol solution extraction, ethyl acetate extraction, column chromatography on silica gel, polyamide column chromatography, methanol dissolution and filtering, separation by efficient preparative liquid chromatography, and product recovery, so that a 4'-O-methyl bavachalcone B monomer product is obtained. According to the invention, through the optimum process and parameter conditions, the content of 4'-O-methyl bavachalcone B in the product is over 98%; and the method is stable in whole technological process, easy to operate, high in separation efficiency, and low in cost, and can realize the preparation of 4'-O-methyl bavachalcone B monomers by massive high-purity separation.
Description
Technical field
The present invention relates to a kind of preparation method of plant compound monomer, especially a kind of separation purification method extracting 4'-O-methyl Bavachalcone B monomer from Psoralea corylifolia, belongs to the separating and purifying technology field of active ingredient of Chinese herbs.
Background technology
Psoralea corylifolia (formal name used at school: Psoralea corylifolia Linn.) has another name called: Fructus Psoraleae, the solid fat of mother-in-law, recklessly fragrant-flowered garlic.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 armpit is raw, has colored 10-30 piece, and corolla is yellow or blue, petal obvious tool lobe handle, vexil obovate, and pod is avette, flower, the fruit phase 7-10 month.Psoralea corylifolia 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 activeconstituents in corylifolia L, there is treatment tetter, antitumor, effect of insecticidal and antibacterial.4'-O-methyl Bavachalcone B be again 4'-O-Methylbroussochalcone B, and molecular formula is C
21h
22o
4, molecular weight is 338.4, and structural formula is as follows:
Through retrieval, the pertinent literature at present for Constituents in Fruits of Psoralea corylifolia L research mainly contains:
1) " Psoralea corylifolia reactive site ethyl acetate extract chemical constitution study " (in December, 2011, " Chongqing traditional Chinese medicine research ", 2nd phase (total 64th phase) 13), the methods such as the just anti-phase silica gel column chromatography of employing and preparative chromatography that disclose are separated, use VU, IR, HPLC-MS,
1hNMR,
13the spectral methods such as CNMR carry out Structural Identification, be divided into obtain 9 compounds, be respectively isopsoralen, psoralene, psoralidine, Bavachalcone, Corylifolin methyl ether, Corylifolinin, Isobavachin, Coryfolin, corylin; Namely Psoralea corylifolia reactive site ethyl acetate extract main component is legumin class and flavones ingredient.
2) " research of Constituents in Fruits of Psoralea corylifolia L " (the 25th volume the 4th phase, in July, 2005, Yunnan Normal University), disclosing extraction and separation method is: dry Psoralea corylifolia seed 1kg, four times are extracted with industrial alcohol cold soaking, leach liquor concentrating under reduced pressure obtains ethanol extraction, dissolve with methyl alcohol (9:1), then extract successively with sherwood oil and chloroform, after chloroform portion recycling design, with 80-100 order silica gel mixed sample, carry out silica gel column chromatography, respectively with sherwood oil: acetone (100:0-0:100), chloroform: methyl alcohol (100:0-0:100) gradient elution, thin-layer chromatography detects, merge into 20 parts, 4th part obtains 4 kinds of amorphous powders respectively through purification by silica gel column chromatography and Preparative TLC chromatography, Bavachalcone is respectively through Structural Identification, bavachromene, pine camphor and p-Hydroxybenzaldehyde.
As can be seen here, in the existing method for Constituents in Fruits of Psoralea corylifolia L research, all do not relate to the extraction and isolation to chemical composition single in Psoralea corylifolia, especially more have no the relevant report of industrial separation purifying 4'-O-methyl Bavachalcone B monomer from Psoralea corylifolia.
Summary of the invention
A kind of easy sharp separation is the object of the present invention is to provide to prepare the method for high-content 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 is separated preparation.
In order to realize foregoing invention object, the technical solution used in the present invention is as follows:
A kind of separation purification method of 4'-O-methyl Bavachalcone B monomer, it is characterized in that with the dry mature fruit of legumes psoraleae for raw material, by ethanolic soln extraction, extraction into ethyl acetate, silica gel column chromatography, polyamide column chromatography, dissolve with methanol filters, high performance preparative liquid chromatography is separated, Product recycling obtains 4'-O-methyl Bavachalcone B monomer product, specifically comprises following processing step:
1) extract
Corylifolia L is ground into the meal of 1 ~ 4mm, is incorporated as the ethanolic soln of quality of medicinal material 8 ~ 10 times of volumes, extract 3 ~ 4 times, each 2 ~ 3 hours, reclaim ethanol, merge filtered liquid;
The concentration of volume percent of described ethanolic soln is 90 ~ 95%;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 ~ 4 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio 5:1 carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract; This step can remove a large amount of impurity and to reach micro-product enriched;
4) polyamide column chromatography
According to medicinal extract quality: solvent volume=1g:5 ~ 10ml, the medicinal extract of step 3) gained is added acetic acid ethyl dissolution, sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, sample polymeric amide is mixed in 45 DEG C of dryings, carry out wash-out with methylene dichloride, column chromatography elutriant 40 DEG C is concentrated into dry, 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 is added dissolve with methanol, filter lysate, namely filtered liquid is the raw material of preparation 4'-O-methyl Bavachalcone B monomer;
6) HPLC preparation
Chromatographic column filler is: C
18filler;
Moving phase is: methanol-water solution V/V=83:17;
Get the filtered liquid sample introduction of step 5) gained, sample size counts 2.0-3.0g/ pin with the dry product weight contained by filtered liquid, carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, determined wavelength is 245nm, collects target compound and prepares solution;
7) Product recycling
The HPLC of step 6) gained is prepared solution, and reclaim methyl alcohol, reddish-orange crystals is separated out from remaining aqueous solution, lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer.
The invention has the advantages 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 easy to concentrated, also reduces energy consumption.
2. the present invention adopts preparative high performance liquid chromatography system to carry out separation and purification 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, be convenient to control quality product, product purity can reach more than 98% simultaneously.
3. because high performance liquid chromatography requires higher to the color and luster, purity etc. of sample, the extracting solution obtained by simple process can not direct injection, if therefore before carrying out high performance liquid chromatography separation, not in accordance with the inventive method step, pre-treatment is carried out to sample introduction solution, then not only separating effect is bad, but also may cause instrument and have a strong impact on (as shortened the life-span etc.), and then increase the production cost of product.
4. for the physico-chemical property of the various compositions existed in corylifolia L, the inventive method is by abovementioned steps 1), 2), 3), 4) order collocation, and suitable parameters combination, the compositions such as 4'-O-methyl Bavachalcone B are effectively extracted, and eliminate a large amount of impurity, obtain thus and can enter the sample solution of preparative high performance liquid chromatography system, unlikely very large impact is caused on highly effective liquid phase chromatographic system, extend its life cycle as far as possible, save production cost.
5., in high performance liquid chromatography sepn process, selection and the combination thereof of each chromatographic condition are very important, because it directly affects the appearance time, peak shape etc. of material; Chromatographic condition mainly comprises chromatographic column (comprising filler, column length, column temperature etc.), moving phase (comprising composition, flow velocity etc.), detector and determined wavelength etc.The present invention, by a large amount of experimental studies and comparative analysis, determines above-described chromatographic condition, thus makes the appearance time of material, peak shape, separating effect etc. reach optimizing, achieves 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 separation and prepare 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.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but this should be interpreted as the present invention is only limitted to following embodiment.
In following each embodiment, the purity of finished product 4'-O-methyl Bavachalcone B monomer is rechecked and is all adopted inverse analysis type liquid chromatography (RP-HPLC) method, and chromatographic condition is as follows:
Filler is C
18, moving phase is methanol-water solution (87:13), and column temperature is 30 DEG C, and flow velocity is 1.0ml/min, and determined wavelength is 245 nm.
embodiment 1
A separation purification method for 4'-O-methyl Bavachalcone B monomer, comprises following processing step:
1) extract
Corylifolia L 45kg is ground into the meal of 1 ~ 4mm, adds the ethanolic soln that 360L concentration of volume percent is 90%, extract 3 times, each 2 hours, reclaim ethanol, merge filtered liquid, altogether 20000ml;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio (5:1) carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract 100 grams;
4) polyamide column chromatography
The medicinal extract of step 3) gained is added 500 ml acetic acid ethyl dissolutions, sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, and sample polymeric amide is mixed in 45 DEG C of dryings, carries out wash-out with methylene dichloride, and column chromatography elutriant 40 DEG C is concentrated into dry, obtains solids 20 grams;
5) dissolution filter
Step 4) gained solids adds 500ml dissolve with methanol, filters lysate, obtains filtered liquid 490ml;
6) HPLC preparation
Employing filler is C
18chromatographic column, post specification is 50cm × 10cm;
Moving phase is: methanol-water solution V/V(87:13);
Determined wavelength is 245nm, ambient operation;
Get the filtered liquid sample introduction of step 5) gained, sample size is 2g/ pin (the dry product weighing scale with contained by filtered liquid), 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 the same), get step 5) gained filtered liquid sample introduction, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to mass spectrometric detection result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatography.
7) Product recycling
The HPLC of step 6) gained prepares solution, and reclaim methyl alcohol, reddish-orange crystals is separated out from remaining aqueous solution, lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer product 8.0 grams.
About 15 days whole Production Flow Chart used times.
Calculating product yield is (8.0/45000) × 100%=0.018%.
By changing mobile phase composition, utilize inverse analysis type liquid chromatography (RP-HPLC) to recheck product purity, recording result is 99.90%.
embodiment 2
A separation purification method for 4'-O-methyl Bavachalcone B monomer, comprises following processing step:
1) extract
Corylifolia L 60kg is ground into the meal of 1 ~ 4mm, adds the ethanolic soln that 500L concentration of volume percent is 95%, extract 3 times, each 2 hours, reclaim ethanol, merge filtered liquid, altogether 15000ml;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio (5:1) carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract 150 grams;
4) polyamide column chromatography
The medicinal extract of step 3) gained is added 1200ml acetic acid ethyl dissolution, and sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, and sample polymeric amide is mixed in 45 DEG C of dryings, carries out wash-out with methylene dichloride, and column chromatography elutriant 40 DEG C is concentrated into dry, obtains solids 30 grams;
5) dissolution filter
Step 4) gained solids adds 600ml dissolve with methanol, filters lysate, obtains filtered liquid 590ml;
6) HPLC preparation
Employing filler is C
18chromatographic column, post specification is 50cm × 10cm;
Moving phase is: methanol-water solution V/V=(87:13);
Determined wavelength is 245nm, ambient operation;
Get the filtered liquid sample introduction of step 5) gained, sample size is 2.5g/ pin (the dry product weighing scale with contained by filtered liquid), 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 the same), get the filtered liquid sample introduction of step 5) gained, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to mass spectrometric detection result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatography.
7) Product recycling
The HPLC of step 6) gained prepares solution, reclaims methyl alcohol, and remaining aqueous solution has a large amount of reddish-orange crystals to separate out, and lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer product 12.0 grams.
About 12 days whole Production Flow Chart used times.
Calculating product yield is (12.0/60000) × 100%=0.02%.
By changing mobile phase composition, utilize inverse analysis type liquid chromatography (RP-HPLC) to recheck product purity, recording result is 99.10%.
embodiment 3
A separation purification method for 4'-O-methyl Bavachalcone B monomer, comprises following processing step:
1) extract
Corylifolia L 80kg is ground into the meal of 1 ~ 4mm, adds the ethanolic soln that 800L concentration of volume percent is 95%, extract 4 times, each 2 hours, reclaim ethanol, merge filtered liquid, altogether 20000ml;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio (5:1) carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract 250 grams;
4) polyamide column chromatography
The medicinal extract of step 3) gained is added 2500ml acetic acid ethyl dissolution, and sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, and sample polymeric amide is mixed in 45 DEG C of dryings, carries out wash-out with methylene dichloride, and column chromatography elutriant 40 DEG C is concentrated into dry, obtains solids 45 grams;
5) dissolution filter
Step 4) gained solids adds 1800ml dissolve with methanol, filters lysate, obtains filtered liquid 1780ml;
6) HPLC preparation
Employing filler is C
18chromatographic column, post specification is 100cm × 20cm;
Moving phase consists of: methanol-water solution V/V(87:13)
Determined wavelength is 245nm, ambient operation;
Get the filtered liquid sample introduction of step 5) gained, sample size is 5.0g/ pin (the dry product weighing scale with contained by filtered liquid), 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 the same), get the filtered liquid sample introduction of step 5) gained, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to mass spectrometric detection result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatography.
7) Product recycling
The HPLC of step 6) gained prepares solution, and reclaim methyl alcohol, reddish-orange crystals is separated out from remaining aqueous solution, lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer product 18.0 grams.
About 12 days whole Production Flow Chart used times.
Calculating product yield is (18.0/80000) × 100%=0.0225%.
By changing mobile phase composition, utilize inverse analysis type liquid chromatography (RP-HPLC) to recheck product purity, recording result is 99.50%.
Claims (6)
1. the separation purification method of a 4'-O-methyl Bavachalcone B monomer, it is characterized in that with the dry mature fruit of legumes psoraleae for raw material, by ethanolic soln extraction, extraction into ethyl acetate, silica gel column chromatography, polyamide column chromatography, dissolve with methanol filters, high performance preparative liquid chromatography is separated, Product recycling obtains 4'-O-methyl Bavachalcone B monomer product, specifically comprises following processing step:
1) extract
Corylifolia L is ground into the meal of 1 ~ 4mm, is incorporated as the ethanolic soln of quality of medicinal material 8 ~ 10 times of volumes, extract 3 ~ 4 times, each 2 ~ 3 hours, reclaim ethanol, merge filtered liquid;
The concentration of volume percent of described ethanolic soln is 90 ~ 95%;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 ~ 4 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio 5:1 carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract;
4) polyamide column chromatography
According to medicinal extract quality: solvent volume=1g:5 ~ 10ml, the medicinal extract of step 3) gained is added acetic acid ethyl dissolution, sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, sample polymeric amide is mixed in 45 DEG C of dryings, carry out wash-out with methylene dichloride, column chromatography elutriant 40 DEG C is concentrated into dry, obtains solids;
5) dissolution filter
According to solids quality: solvent volume=1g:20 ~ 40ml, step 4) gained solids is added dissolve with methanol, filter lysate, namely filtered liquid is the raw material of preparation 4'-O-methyl Bavachalcone B monomer;
6) HPLC preparation
Chromatographic column filler is: C
18filler;
Moving phase is: methanol-water solution V/V=83:17;
Get the filtered liquid sample introduction of step 5) gained, sample size counts 2.0-3.0g/ pin with the dry product weight contained by filtered liquid, carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, determined wavelength is 245nm, collects target compound and prepares solution;
7) Product recycling
The HPLC of step 6) gained is prepared solution, and reclaim methyl alcohol, reddish-orange crystals is separated out from remaining aqueous solution, lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer.
2. the separation purification method of a kind of 4'-O-methyl Bavachalcone B monomer according to claim 1, is characterized in that comprising following processing step:
1) extract
Corylifolia L 45kg is ground into the meal of 1 ~ 4mm, adds the ethanolic soln that 360L concentration of volume percent is 90%, extract 3 times, each 2 hours, reclaim ethanol, merge filtered liquid, altogether 20000ml;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio 5:1 carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract 100 grams;
4) polyamide column chromatography
The medicinal extract of step 3) gained is added 500 ml acetic acid ethyl dissolutions, sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, and sample polymeric amide is mixed in 45 DEG C of dryings, carries out wash-out with methylene dichloride, and column chromatography elutriant 40 DEG C is concentrated into dry, obtains solids 20 grams;
5) dissolution filter
Step 4) gained solids adds 500ml dissolve with methanol, filters lysate, obtains filtered liquid 490ml;
6) HPLC preparation
Employing filler is C
18chromatographic column, post specification is 50cm × 10cm;
Moving phase is: methanol-water solution V/V=87:13;
Determined wavelength is 245nm, ambient operation;
Get the filtered liquid sample introduction of step 5) gained, sample size counts 2g/ pin with the dry product weight contained by filtered liquid, 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, and reclaim methyl alcohol, reddish-orange crystals is separated out from remaining aqueous solution, lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer product 8.0 grams;
About 15 days whole Production Flow Chart used times;
Calculating product yield is 8.0/45000 × 100%=0.018%;
By changing mobile phase composition, utilize inverse analysis type liquid chromatography RP-HPLC to recheck product purity, recording result is 99.90%.
3. the separation purification method of a kind of 4'-O-methyl Bavachalcone B monomer according to claim 1, is characterized in that comprising following processing step:
1) extract
Corylifolia L 60kg is ground into the meal of 1 ~ 4mm, adds the ethanolic soln that 500L concentration of volume percent is 95%, extract 3 times, each 2 hours, reclaim ethanol, merge filtered liquid, altogether 15000ml;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio 5:1 carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract 150 grams;
4) polyamide column chromatography
The medicinal extract of step 3) gained is added 1200ml acetic acid ethyl dissolution, and sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, and sample polymeric amide is mixed in 45 DEG C of dryings, carries out wash-out with methylene dichloride, and column chromatography elutriant 40 DEG C is concentrated into dry, obtains solids 30 grams;
5) dissolution filter
Step 4) gained solids adds 600ml dissolve with methanol, filters lysate, obtains filtered liquid 590ml;
6) HPLC preparation
Employing filler is C
18chromatographic column, post specification is 50cm × 10cm;
Moving phase is: methanol-water solution V/V=87:13;
Determined wavelength is 245nm, ambient operation;
Get the filtered liquid sample introduction of step 5) gained, sample size counts 2.5g/ pin with the dry product weight contained by filtered liquid, 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 methyl alcohol, and remaining aqueous solution has a large amount of reddish-orange crystals to separate out, and lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer product 12.0 grams;
About 12 days whole Production Flow Chart used times;
Calculating product yield is 12.0/60000 × 100%=0.02%;
By changing mobile phase composition, utilize inverse analysis type liquid chromatography RP-HPLC to recheck product purity, recording result is 99.10%.
4. the separation purification method of a kind of 4'-O-methyl Bavachalcone B monomer according to claim 1, is characterized in that comprising following processing step:
1) extract
Corylifolia L 80kg is ground into the meal of 1 ~ 4mm, adds the ethanolic soln that 800L concentration of volume percent is 95%, extract 4 times, each 2 hours, reclaim ethanol, merge filtered liquid, altogether 20000ml;
2) extract
By step 1) filtered liquid of gained, add isopyknic extraction into ethyl acetate 3 times, point get upper solution, be concentrated into dry, 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 sample silica gel is mixed in 45 DEG C of dryings, with sherwood oil: ethyl acetate volume ratio 5:1 carries out wash-out, and column chromatography elutriant 45 DEG C is concentrated into dry, obtains medicinal extract 250 grams;
4) polyamide column chromatography
The medicinal extract of step 3) gained is added 2500ml acetic acid ethyl dissolution, and sample mixed by the polymeric amide that the solution of gained adds 4 times of volumes, and sample polymeric amide is mixed in 45 DEG C of dryings, carries out wash-out with methylene dichloride, and column chromatography elutriant 40 DEG C is concentrated into dry, obtains solids 45 grams;
5) dissolution filter
Step 4) gained solids adds 1800ml dissolve with methanol, filters lysate, obtains filtered liquid 1780ml;
6) HPLC preparation
Employing filler is C
18chromatographic column, post specification is 100cm × 20cm;
Moving phase consists of: methanol-water solution V/V=87:13
Determined wavelength is 245nm, ambient operation;
Get the filtered liquid sample introduction of step 5) gained, sample size counts 5.0g/ pin with the dry product weight contained by filtered liquid, carries out the preparative separation of 4'-O-methyl Bavachalcone B monomer, online ultraviolet monitoring, and collection obtains target compound and prepares solution 25000ml;
7) Product recycling
The HPLC of step 6) gained prepares solution, and reclaim methyl alcohol, reddish-orange crystals is separated out from remaining aqueous solution, lets cool filtration and namely obtains 4'-O-methyl Bavachalcone B monomer product 18.0 grams;
About 12 days whole Production Flow Chart used times;
Calculating product yield is 18.0/80000 × 100%=0.0225%;
By changing mobile phase composition, utilize inverse analysis type liquid chromatography RP-HPLC to recheck product purity, recording result is 99.50%.
5., according to the separation purification method of a kind of 4'-O-methyl Bavachalcone B monomer in claim 1-4 described in any one, it is characterized in that: the chromatographic condition that described inverse analysis type liquid chromatography RP-HPLC carries out purity reinspection is as follows:
Filler is C
18, moving phase is methanol-water solution V/V=87:13, and column temperature is 30 DEG C, and flow velocity is 1.0ml/min, and determined wavelength is 245nm.
6. according to the separation purification method of a kind of 4'-O-methyl Bavachalcone B monomer in claim 1-4 described in any one, it is characterized in that: before carrying out high performance preparative liquid chromatography separation, by the peak shape of 4'-O-methyl Bavachalcone B monomer in liquid-mass chromatography method determination high performance liquid chromatography, namely step 5) gained filtering solution sample introduction is got, carry out the preparative separation of 4'-O-methyl Bavachalcone B monomer, according to mass spectrometric detection result, determine the peak shape that 4'-O-methyl Bavachalcone B monomer is corresponding in liquid chromatography.
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