CN109988133A - A kind of preparation method of danshinolic acid Y - Google Patents
A kind of preparation method of danshinolic acid Y Download PDFInfo
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Abstract
The present invention relates to the preparation methods of danshinolic acid Y a kind of, it the described method comprises the following steps: the enrichment of step 1, danshinolic acid Y: taking root of red-rooted salvia phenolic acid extract, it is dissolved with water, be added in MCI-GEL chromatographic column, with 10-20% ethanol water elute, under connect liquid adjust pH value, it is added in MCI-GEL chromatographic column again, with 70-95% ethanol elution, recycling design is concentrated to dryness to obtain danshinolic acid Y enriched substance；Step 2, fine separation: danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 1-3 times of quality volume, it is pressurizeed using dynamic axial and prepares liquid phase separation, mobile phase is acetonitrile and the aqueous solution containing 0.01% formic acid, the eluent containing danshinolic acid Y is detected and merged with HPLC method, is concentrated under reduced pressure to give danshinolic acid Y monomer.
Description
Technical field
The present invention relates to a kind of field of medicine and chemical technology, and in particular to a kind of preparation method of danshinolic acid Y.
Background technique
Danshinolic acid Y (salvianolic acid Y), chemical name are (2R, 3S) -4- [(1E) -3- [(1R) -1- carboxyl -
2- (3,4- dihydroxy phenyl) ethyoxyl] -3- oxygen -1- propylene -1- base] -2- (3,4- dihydroxy phenyl) -2,3- dihydro -7- hydroxyl
Base -3- benzofuran base formic acid-[(1R) -1- carboxyl -2- (3,4- dihydroxy phenyl)] ethyl ester, is from salvia root polyphenol acid extract
In isolated tanshin polyphenolic acid B an epimer, there is stronger effect of scavenging radical, structure is shown in formula I:
CN105218495A (application No. is 201410226328.5) discloses danshinolic acid Y and preparation method thereof, preparation
Method are as follows: red rooted salvia adds water to cook extraction 3 times, and every time plus 3-6 times is measured water, decocts 0.5-2 hours, collecting decoction is cooled to
8-14 DEG C, with hydrochloric acid solution tune pH value to 1.0-2.0,4-8 hours is stood, supernatant is taken, filtered；Filtrate crosses polyamide column, with 7
The purified water of times column volume is rinsed, and discards efflux, then with the 0.1% sodium bicarbonate solution elution of 7 times of column volumes, is collected and eluted
Liquid, with hydrochloric acid solution tune pH value to 2.5-3.5；AB-8 type large pore resin absorption column is crossed, is rushed with the purified water of 2-3 times of column volume
It washes, discards water lotion, then with about 2.5 times of 95% ethanol elutions of column volume, collect eluent, ethyl alcohol is recovered under reduced pressure, is adjusted to 30
Relative density 1.02-1.06 under the conditions of DEG C, 2-10 DEG C refrigeration 12-24 hours, filtration, filtrate is with 10% sodium hydroxide solution tune pH
Value to 5.0-6.5, freeze-drying obtains extract, extract is dissolved with suitable quantity of water, is purified by half preparation RP-HPLC twice,
Obtain sterling.
Root of red-rooted salvia phenolic acid extract, commercially available salvianolic acid the extract (" China including meeting 2015 editions Chinese Pharmacopoeia standards
Pharmacopeia " 2015 years the 1st middle 397-398 pages of versions) and existing market on the preparation raw material of Danshen injection Polyphenol Acids sold it is red
Joining Polyphenol Acids extract, (its method is a variety of, such as CN105588885A, and application No. is in 201410572435.3 background techniques
The preparation method mentioned), wherein meeting the commercially available salvianolic acid method for preparing extractive of 2015 editions Chinese Pharmacopoeia standards are as follows: take
Radix Salviae Miltiorrhizae is cut into segment, and water is added to extract in 80 DEG C twice, combined extract, filtration, and filtrate is concentrated under reduced pressure into relative density in 60 DEG C
It for the medicinal extract of 1.18-1.22 (50 DEG C), lets cool, adds ethyl alcohol that alcohol content is made to be 70%, stand 12 hours, take supernatant, depressurize
Recycle ethyl alcohol, and be concentrated into thick paste, it is dry to get.
Find that the method for building up of root of red-rooted salvia phenolic acid extract finger-print is shown in experimental example 2, root of red-rooted salvia phenolic acid by further analysis
The finger-print of extract is shown in that Fig. 1, root of red-rooted salvia phenolic acid extract include protocatechualdehyde, salvianolic acid D, tanshin polyphenolic acid B, Rosmarinic acid, pellet
Phenolic acid Y, alkannic acid and (7'S, 8'R, 8 " R)-epi- alkannic acid etc..Salvianolic acid extract and Danshen injection Polyphenol Acids
The TuPu method and shared peak of both raw materials (i.e. salvia root polyphenol acid extract) are consistent.
A kind of ingredient of the danshinolic acid Y as root of red-rooted salvia phenolic acid extract has good pharmaceutical activity and important Radix Salviae Miltiorrhizae group
Point, the danshensu Y for obtaining high-purity can prepare standard substance, new drug research can also be carried out, and obtain in the prior art
The method for obtaining danshensu Y, with high costs, purity is not high, the wasting of resources, for this reason, it may be necessary to improve efficiency.The present invention provides one kind
The method of isolated danshinolic acid Y is extracted in new slave root of red-rooted salvia phenolic acid extract.
Summary of the invention
The present invention provides the preparation method of danshinolic acid Y a kind of, and the method is from root of red-rooted salvia phenolic acid extract by danshinolic acid Y
It carries out isolated, for this purpose, the present invention provides the preparation method of danshinolic acid Y a kind of, the described method comprises the following steps:
The enrichment of step 1, danshinolic acid Y
Root of red-rooted salvia phenolic acid extract is taken, is dissolved with water, is added in MCI-GEL chromatographic column, with 10-20% ethanol aqueous wash
It is de-, under connect liquid and adjust pH value, MCI-GEL chromatography column solid phase extraction is used again, with 70-95% ethanol elution, recycling design, concentration
Danshinolic acid Y enriched substance is obtained to dry；
Step 2, fine separation
Danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 1-3 times of quality volume, is pressurizeed using dynamic axial and is prepared liquid phase
Separation, mobile phase are acetonitrile and the aqueous solution containing 0.01% formic acid, are detected with HPLC method and merge the elution containing danshinolic acid Y
Liquid is concentrated under reduced pressure to give danshinolic acid Y monomer.
In the above method:
In the step 1:
Root of red-rooted salvia phenolic acid extract is the salvianolic acid extract prepared according to official method, or according to prior art preparation
Danshen injection Polyphenol Acids raw material or Radix Salviae Miltiorrhizae water extract.
The pH value of the root of red-rooted salvia phenolic acid solution of extract is that 5.0-6.5 needs to adjust if its pH value is not in this range
Section is 5.0-6.5；
Cross column twice, the ratio of first time extract quality and packing volume is 1:60-1:20, second of extract quality with
The ratio of packing volume is 1:3-1:10, and same root chromatogram column can be used twice, with two same sizes and can also be filled out
The chromatographic column of material.
Adjust pH value to 2.5-4, preferably 2.8-3.2；
The regulator for adjusting pH value is formic acid or hydrochloric acid.
The volume ratio of mobile phase acetonitrile and the aqueous solution containing 0.01% formic acid is 23-25:77-75 in step 2.
In step 1-3 of the present invention, the collection of ingredient, respectively be with HPLC method detect, and with root of red-rooted salvia phenolic acid extract fingerprint
Map (see Fig. 1, method for building up is shown in experimental example 2) control, collects the eluent containing danshinolic acid Y.
Preferably, the preparation method comprises the following steps:
The enrichment of step 1, danshinolic acid Y: taking root of red-rooted salvia phenolic acid extract, dissolved with water, is added in MCI-GEL chromatographic column,
With 10-20% ethanol water elute, under connect liquid adjust pH value, be added in MCI-GEL chromatographic column again, with 70-95% second
Alcohol elution, recycling design are concentrated to dryness to obtain danshinolic acid Y enriched substance；
Step 2, fine separation: danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 1-3 times of quality volume, using dynamic
Axial pressure prepares liquid phase separation, and mobile phase is acetonitrile and the aqueous solution containing 0.01% formic acid, is detected and is merged with HPLC method and contains
There is the eluent of danshinolic acid Y, is concentrated under reduced pressure to give danshinolic acid Y monomer.
The present invention further comprises the step of step 2 gained danshinolic acid Y monomer is dried, and preferably 60 DEG C are dried under reduced pressure
10-15 hours.
Further preferably, the preparation method comprises the following steps:
The enrichment of step 1, danshinolic acid Y: taking root of red-rooted salvia phenolic acid extract, is dissolved with the 1-3 times of water measured, and MCI Gel color is added
It composes in column, the ratio of extract quality and chromatographic column filler volume is 1:60-1:20, and loading speed is 1-3BV/h, then uses 10-
The elution of 20% ethanol water, flow velocity 1-3BV/h are detected with HPLC method, and compareed with root of red-rooted salvia phenolic acid extract finger-print,
The eluent containing danshinolic acid Y is collected, pH value 2.5-4 is adjusted, again with the absorption enrichment of MCI Gel chromatographic column, original extract
The ratio of quality and chromatographic column filler volume is 1:3-1:10, and with the water elution of 3-5BV, depickling is discarded, and finally uses 70-95% second
Alcohol elution, flow velocity 1-3BV/h collect eluent, are concentrated to dryness to obtain danshinolic acid Y enriched substance；
Step 2, fine separation: danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 1-3 times of quality volume, using dynamic
Axial pressure prepares liquid phase separation, each sample introduction mobile phase: acetonitrile -0.01% aqueous formic acid 23-25:77-75, flow velocity 5-
7BV/h detects with HPLC method and merges the eluent containing danshinolic acid Y, is concentrated under reduced pressure to give danshinolic acid Y monomer；
Still more preferably, the preparation method comprises the following steps:
The enrichment of step 1, danshinolic acid Y: taking root of red-rooted salvia phenolic acid extract, and MCIGel color is added in the water dissolution of 2 times of quality volumes
It composes in column, each loading extracts ratio 1:60~1:20 of object amount-MCI-Gel packing volume, flow velocity 2BV/h, uses after end of the sample
The elution of 10%~20% ethanol water, flow velocity 2BV/h adjust eluent pH value to 2.8-3.2, be added again it is regenerated with
In upper MCI Gel chromatographic column, 5BV, depickling being washed with water, eluent discards, flow velocity: 2BV/h is washed with 80-95% ethyl alcohol later
De-, flow velocity: 2BV/h collects eluent 2BV, and recycling design is concentrated to dryness to obtain danshinolic acid Y enriched substance；
Step 2, fine separation: danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 2 times of quality volumes, using dynamic shaft
Liquid phase separation is prepared to pressurization, each sample introduction mobile phase: acetonitrile -0.01% aqueous formic acid 23-25:77-75, flow velocity 6BV/h,
Every 1/4BV connects a sample, and the eluent containing danshinolic acid Y is detected and merged with HPLC method, it is mono- to be concentrated under reduced pressure to give danshinolic acid Y
Body；
It is the explanation to vocabulary of terms below:
Depickling, the fraction collected on first pass MCI is eluted with water after being added again in MCI-GEL column both can will be extra
Pickling take off, retain danshinolic acid Y, use ethanol elution later, then be concentrated, the increasing of concentration process acid concentration will be avoided to cause
Degradation.
BV/h is bed volume/hour, for example 2BV/h is 2 times of bed volume/hours；
BV is bed volume, for example 1/10BV is 1/10 bed volume；
Regeneration: MCI chromatographic column after first pass use 95% ethanol elution of 5BV, then the water elution of 5BV is used, completion
Regeneration is used for second time Solid Phase Extraction.
Method provided by the invention has the advantage that
1, danshinolic acid Y belongs to phenolic acid compound, and phenolic hydroxyl group is more in structure, is separated and is extracted using resin
When, such as the method in CN105218495A (application No. is 201410226328.5, abbreviation danshinolic acid Y compound patent) patent,
Phenolic hydroxyl group is eluted in conjunction with the amide groups in polyamide with sodium bicarbonate solution, is formed sodium salt and is eluted, uses hydrochloric acid later
Solution adjusts pH value, and phenolate is made to be converted into free state phenolic acid, to preferably be absorbed by macroporous absorbent resin, also therefore, adjusts
It is the step essential, indispensable using resin adsorption at acidity.Meanwhile patented method danshinolic acid Y in order to obtain, also
Using 10% sodium hydroxide solution tune pH value to 5.0-6.5, during this, phenolic acid class is easy to react with highly basic, in and
Hydrochloric acid while, and some danshinolic acid Y is degraded.Again by half preparation RP-HPLC purifying twice, sterling is obtained.
Inventor carries out enrichment discovery using the above method, is only by danshinolic acid Y enriched substance content prepared by resin column
1.0% or so, and using half preparation RP-HPLC purifying, workload is very big, and preparation 100mg needs 5-10 days, and difficult to realize
Amplification preparation.
In order to which danshinolic acid Y to be enriched to certain purity (30-50%), inventor has been selected using positive phase filling for being enriched with
Danshinolic acid Y has primarily determined scheme such as reference examples 1, it may be assumed that first uses silica gel post separation, then is divided with ODS (C18 filler) chromatographic column
From last available a small amount of danshinolic acid Y.By the isolated intermediate of normal phase silica gel chromatography, danshinolic acid Y normalization percentage contains
Amount can achieve 50% or more, and yield is also increased slightly, but this method separation cycle is long, and the organic solvent used has acetic acid second
Ester, petroleum ether, acetonitrile etc., in addition liposoluble ingredient is more close in conjunction with silicone hydroxyl on a silica gel column, cause hangover serious and
It is unfavorable for separating, therefore also needs to prevent from trailing using organic acid, experimental studies have found that, first is added in solvent through TLC
For the amount of acid when reaching 10%, the thin layer spot of danshinolic acid Y could become round spot from vertical bar band spot, through groping with it is appropriate
It assists, 2% formic acid is finally used in reference examples.Safety and environment of the use of a large amount of organic solvents and organic acid to operator
Bring hidden danger.
Inventor not will use excessive acid, without repeatedly the study found that when preparing using reverse chromatograms column
The pH value for adjusting intermediate feed liquid reduces degradation loss.
In order to solve problem above.Inventor is by screening, and finally using MCI-GEL separation, (mobile phase uses low concentration
Ethanol water, be not necessarily to acid adding) obtain the eluent containing danshinolic acid Y, ethanol water of the danshinolic acid Y in low concentration in order to prevent
It is decomposed when being concentrated in solution, inventor reuses MCI-GEL solid phase extraction and is enriched with to obtain the high concentration second of a small amount of danshinolic acid Y
The eluent of alcohol can obtain the crude product of danshinolic acid Y using short period concentration.
Prior art such as CN102241574A (application No. is 201010169711.3) discloses a kind of system of protocatechualdehyde
Preparation Method, which use MCI-GEL separation, raw material is the Radix Salviae Miltiorrhizae extractum that alkali carries take alcohol precipitation, and then chromatography is gone polar
Small Components in Salvia miltiorrhiza, chromatography separate protocatechualdehyde, and deacidify decoloration, polishing purification, drying.With danshinolic acid Y compound patent phase
Seemingly, it is adsorbed with macroporous absorbent resin, has also used a large amount of acid.
Compared with prior art with the method for screening, operate in this way, can to avoid using using for organic solvent and organic acid,
The decomposition of danshinolic acid Y most importantly can be effectively prevented, improve yield.The crude product of danshinolic acid Y reuses ODS purifies and separates and obtains
To the sterling of danshinolic acid Y.
2, after the enriched substance for obtaining danshinolic acid Y, in order to preferably separate with unknown impuritie (see Fig. 2), inventor is by touching
Rope is found to be separated by the way of manual pressure using the C18 filler of 50 micron grain sizes, is extremely difficult to separate, and flow velocity is not
Ideal, separation cycle are very slow.Finally selection separating degree and the axial pressure preparative chromatograph of HPLC relatively, and by HPLC
Method screens mobile phase.The results show that the methanol of different proportion: water (0.02% formic acid) separating degree is unsatisfactory, and 20%~
25% -0.01% aqueous formic acid separating effect of acetonitrile is preferable, and practice to dynamic axial is pressurizeed in preparation chromatography, 23% second
- 0.01% aqueous formic acid of nitrile is ideal ratio.
3, the prior art is isolated few using polyamide, large pore resin absorption column, 2 ODS-HPLC preparative liquid chromatographies
Danshinolic acid Y is measured, this method is appropriate only for the preparation of mg grades of samples, can be used for compound identification demand etc..If necessary into one
Step studies the pharmacological activity etc. of the compound, needs g grades of samples, the prior art then cannot achieve.
4, the present invention uses root of red-rooted salvia phenolic acid for raw material, separates by 2 step reverse phase fillers, available 98.5% or more pellet
Phenolic acid Y monomer, step is few, purity is high, easy to operate, can be used for the exploitation of cardio-cerebrovascular new drug.
Detailed description of the invention
Fig. 1: the finger-print of root of red-rooted salvia phenolic acid extract；
The HPLC of Fig. 2: danshinolic acid Y enriched substance (lot number 20140530) detects figure；
The HPLC of Fig. 3: danshinolic acid Y monomer (lot number 20140620) detects figure.
Specific embodiment
Drug of the invention is described further below with reference to embodiment, following each embodiments are merely to illustrate the present invention
And not limitation of the present invention.
A kind of embodiment 1: preparation method of danshinolic acid Y
The enrichment of step 1, danshinolic acid Y
Root of red-rooted salvia phenolic acid extract 500g is taken, with 1000ml water (w/v of extract and water is 1:2) dissolution, uses pH
Meter detection pH value be 5.5, each loading 100ml, addition be filled in 3L MCI Gel glass column (extract quality with fill out
Material volume ratio is 1:60), flow velocity: 100ml/min.It is eluted after end of the sample with 10% ethanol water, flow velocity: 100ml/min,
Every 300ml connects a sample connecing liquid under and having color, is detected with HPLC method, at the same with root of red-rooted salvia phenolic acid extract finger-print pair
According to collecting and merge the eluent containing danshinolic acid Y.
Eluent pH value is adjusted to 3.0, be added again be filled with 3LMCI Gel glass column (extract quality with fill out
Material volume ratio is 1:6) in, 15L is washed with water, eluent discards, flow velocity: 100ml/min.Later with 95% ethanol elution, stream
Speed: 100ml/min collects eluent 6L, and recycling design is concentrated to dryness to obtain danshinolic acid Y enriched substance.Operation repetitive above step
The separation for completing all samples, is obtained enriched substance 24g, lot number 20140530.
Step 2, fine separation
Danshinolic acid Y enriched substance 20g is completely dissolved with 40ml mobile phase, is pressurizeed using dynamic axial and is prepared liquid phase separation, often
Secondary sample introduction 10ml, mobile phase: the solution 23:77 of -0.01% formic acid of acetonitrile, flow velocity 200ml/min, every 500ml connect a sample.
It is detected with HPLC method, while being compareed with the finger-print of root of red-rooted salvia phenolic acid extract, merge the eluent containing danshinolic acid Y.Decompression
It is concentrated to get danshinolic acid Y monomer, the separation of all samples is completed in the parallel above operation, obtains 4.1g danshinolic acid Y, normalization method hundred
Dividing content is 99.5%, lot number 20140620.
Step 3, drying
60 DEG C are dried under reduced pressure 12 hours.
Embodiment 2: the expansion preparation method of danshinolic acid Y
The enrichment of step 1, danshinolic acid Y
Root of red-rooted salvia phenolic acid extract 1000g is taken, is dissolved with 2000ml water, is 5.5 with pH meter detection pH value, each loading
100ml, addition are filled in 3L MCI Gel glass column (extract quality is 1:60 with packing volume ratio), flow velocity:
100ml/min.It is eluted after end of the sample with 10% ethanol water, flow velocity: 100ml/min, it is every under connecing liquid and having color
300ml connects a sample.It is detected with HPLC method, while being compareed with the finger-print of root of red-rooted salvia phenolic acid extract, merge 15-25 bottles
Eluent containing danshinolic acid Y.
Eluent pH value is adjusted to 3.0, be added again be filled with 3L MCI Gel glass column (extract quality with fill out
Material volume ratio is 1:3) in, 15L is washed with water, eluent discards, flow velocity: 100ml/min.Later with 95% ethanol elution, stream
Speed: 100ml/min collects eluent 6L, and recycling design is concentrated to dryness to obtain danshinolic acid Y enriched substance.Operation repetitive above step
The separation for completing all samples, is obtained enriched substance 35g, lot number 20150604.
Step 2, fine separation
Danshinolic acid Y enriched substance is completely dissolved with 35g with 70ml mobile phase, is pressurizeed using dynamic axial and is prepared liquid phase separation,
Each sample introduction 10ml, mobile phase: -0.01% aqueous formic acid 23:77 of acetonitrile, flow velocity 200ml/min, every 500ml connect a sample
Product.It is detected with HPLC method, while being compareed with the finger-print of root of red-rooted salvia phenolic acid extract, merge the eluent containing danshinolic acid Y.Subtract
Pressure is concentrated to get danshinolic acid Y monomer, and the separation of all samples is completed in the parallel above operation, obtains 10.3g danshinolic acid Y, normalizes
Method percentage composition is 99.5%, lot number 20151113.
Step 3, drying
60 DEG C are dried under reduced pressure 12 hours.
Experimental example 1: the assay of danshinolic acid Y
1, using high performance liquid chromatography, actual conditions are as follows
Instrument: 1100 high performance liquid chromatograph of Agilent, quaternary pump, UV detector.
Chromatographic column: Agilent C18 analytical column (4.6mm × 250mm, 5 μm)；Mobile phase: gradient elution is used, is shown in Table 1；
Flow velocity: 1mLmin-1；Detection wavelength 280nm；Column temperature: 30 DEG C.
1 chromatographic condition gradient of table
| Solvent time (min) | 0 | 8 | 15 | 35 | 40 | 50 |
| A:80% acetonitrile+20%B | 10% | 22% | 26% | 39% | 10% | 10% |
| B:0.02% phosphate aqueous solution | 90% | 78% | 74% | 61% | 90% | 90% |
2, the preparation of testing sample solution: taking danshinolic acid Y10mg, accurately weighed, sets in 25ml volumetric flask, molten with methanol
Solution, and it is dissolved to scale, it is to be measured.
3, assay: each 10 μ L of sample solution is taken, is measured, each sample is in parallel into two needles.
4, assay is as a result, be shown in Table 2, Fig. 2 and Fig. 3.
2 danshinolic acid Y sample size measurement result of table
| Sample ID | Percentage composition (%) |
| Danshinolic acid Y enriched substance 20140530 | 21% |
| Danshinolic acid Y enriched substance 20150620 | 22% |
| High-purity danshinolic acid Y20140604 | 99.5% |
| High-purity danshinolic acid Y20151113 | 98.4% |
Comparative example 1: screening experiment
Step 1: the silica gel column chromatography of danshinolic acid Y separates
Root of red-rooted salvia phenolic acid extract 100g is taken, is dissolved in 200ml water, is extracted 3 times with isometric ethyl acetate, acetic acid is collected
Solvent is recovered under reduced pressure in ethyl ester extract liquor, and the 180g 100-200 mesh silica gel that raw material is added carries out mixing sample.
Silicagel column loads: taking the 200-300 mesh silica gel of raw material 1000g, is soaked with ethyl acetate-light petrol 1:1 (2% formic acid)
30min is steeped, column is filled, balances 3BV with ethyl acetate-light petrol 1:1.
Separation: sample dry method loading is eluted with ethyl acetate-light petrol 1:1 (2% formic acid), and about 0.2BV is unit receipts
Collect fraction, TLC detection, is that color developing agent develops the color with 5% sulfuric acid ethyl alcohol.
Fraction merges: will merge with the consistent fraction of danshinolic acid Y reference substance spot and R f value, and be concentrated to dryness, contained
There is the crude product 1.83g of danshinolic acid Y.
Step 2: fine separation
Danshinolic acid Y crude product 1.83g is completely dissolved with 23% acetonitrile solution of 10ml, using dynamic axial pressurization preparation solution
Phase (being filled with reverse phase C18 filler) separation, each sample introduction 10ml, mobile phase: -0.01% aqueous formic acid 23:77 of acetonitrile, flow velocity
200ml/min, every 500ml connect a sample.It is detected with HPLC method, while being compareed with the finger-print of root of red-rooted salvia phenolic acid extract,
Merge the eluent containing enriched substance.It is concentrated under reduced pressure to give danshinolic acid Y 0.4g.
Using the available danshinolic acid Y of this method, but a collection of long preparation period, probably need 10 days；Next was prepared
A large amount of organic reagents and organic acid are used in journey, cause ratio to influence preparation personnel safety and environment；In addition, since the later period is dense
Since the concentration of acid continues to increase when contracting, lead to the danshinolic acid Y fast degradation in eluent, so that yield is very low.Comparative example 2:
With reference to the method for CN102212003A (application No. is 201010143666.4) patent disclosure, difference is to collect red
Phenolic acid Y (is monitored) by the method for experimental example 2.
Salvia root polyphenol acid 100g is taken, aqueous solution is added in MCI-GEL (3L MCI-GEL) column, with 20% ethanol aqueous wash
It is de-, sample is connect since dripping under color band, every 300ml connects a fraction, meets 2.5L in total, then eluted with 35% ethanol solution, altogether
Collect 6L.
By contrasting detection, for first colour band in totally 9 bottles of collection liquids, danshinolic acid Y is concentrated mainly on 1-3 bottles, with pigment and
A large amount of impurity are simultaneously deposited.
Danshinolic acid Y in 1-3 bottles is separated, discovery due to being unable to reach concentration effect, will not continue into
Row separates in next step.
Experimental example 2: the detection of each ingredient in existing root of red-rooted salvia phenolic acid extract
1, use method for ultra performance liquid chromatography (Ultra Performance Liquid Chromatography,
UPLC) method, specific chromatographic condition are as follows:
Instrument: Waters UPLC-PDA
Chromatographic column: Waters Acquity HSS T3 1.8um
Mobile phase: A:0.1% formic acid water, B: acetonitrile
Flow velocity: 0.4mLmin-1；Detection wavelength 280nm；Column temperature: 30 DEG C.
Gradient elution is shown in Table 3:
Table 3: gradient elution table
| T(min) | A (%) | B (%) |
| 0 | 93 | 7 |
| 6.4 | 86 | 14 |
| 16.0 | 84 | 16 |
| 22.0 | 83 | 17 |
| 28.0 | 82 | 18 |
| 32.0 | 78 | 22 |
| 35.0 | 20 | 80 |
| 35.5 | 93 | 7 |
| 40 | 93 | 7 |
2, the preparation method of test solution:
Root of red-rooted salvia phenolic acid extract 50mg is weighed, sets in 25ml volumetric flask, is dissolved with methanol, and be dissolved to scale, it is to be measured.
3, it detects: taking 2 μ L of sample solution, sample introduction measurement.
The result is shown in Figure 1 contains protocatechualdehyde, salvianolic acid D, tanshin polyphenolic acid B, Rosmarinic acid, red phenol in root of red-rooted salvia phenolic acid extract
Sour Y, alkannic acid and (7'S, 8'R, 8 " R)-epi- alkannic acid, the raw material of salvianolic acid extract and Danshen injection Polyphenol Acids
The map and shared peak of both (i.e. salvia root polyphenol acid extract) are consistent.
Claims (10)
1. a kind of preparation method of danshinolic acid Y, which is characterized in that the described method comprises the following steps:
The enrichment of step 1, danshinolic acid Y
Root of red-rooted salvia phenolic acid extract is taken, is dissolved with water, is added in MCI-GEL chromatographic column, is eluted with 10-20% ethanol water, under
It connects liquid and adjusts pH value, use MCI-GEL chromatography column solid phase extraction again, with 70-95% ethanol elution, recycling design is concentrated to dryness
Obtain danshinolic acid Y enriched substance；
Step 2, fine separation
Danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 1-3 times of quality volume, is pressurizeed using dynamic axial and is prepared liquid phase point
From, mobile phase is acetonitrile and the aqueous solution containing 0.01% formic acid, the eluent containing danshinolic acid Y is detected and merged with HPLC method,
It is concentrated under reduced pressure to give danshinolic acid Y monomer.
2. preparation method according to claim 1, which is characterized in that the step 1, wherein the root of red-rooted salvia phenolic acid extracts
Object is dissolved with water, and the pH value of solution, which needs to adjust, arrives 5.0-6.5.
3. preparation method according to claim 1, which is characterized in that the step 1, wherein be added to MCI-GEL twice
In chromatographic column, the ratio of first time extract quality and packing volume is 1:60-1:20, second of extract quality and packing volume
Ratio be 1:3-1:10.
4. preparation method according to claim 1, which is characterized in that the step 1, wherein connect liquid under described and adjust pH
Value adjusts pH value to 2.5-4, preferably 2.8-3.2；The regulator for adjusting pH value is formic acid or hydrochloric acid.
5. preparation method according to claim 1, which is characterized in that the step 1 mobile phase acetonitrile and contains in step 2
The volume ratio of the aqueous solution of 0.01% formic acid is 23-25:77-75.
6. preparation method according to claim 1-5, which is characterized in that method includes the following steps:
The enrichment of step 1, danshinolic acid Y: taking root of red-rooted salvia phenolic acid extract, dissolved with water, is added in MCI-GEL chromatographic column, uses 10-
20% ethanol water elution, under connect liquid adjust pH value, be added in MCI-GEL chromatographic column, washed with 70-95% ethyl alcohol again
De-, recycling design is concentrated to dryness to obtain danshinolic acid Y enriched substance；
Step 2, fine separation: danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 1-3 times of quality volume, using dynamic axial
Pressurization preparation liquid phase is separated, and obtains the eluent containing danshinolic acid Y, it is 23- that the mobile phase eluent used, which is volume ratio,
The mixture of the acetonitrile of 25:77-75 and the aqueous solution containing 0.01% formic acid is dense by being concentrated and dried containing the eluent of danshinolic acid Y
Contracting obtains danshinolic acid Y monomer.
7. preparation method according to claim 6, which is characterized in that the described method comprises the following steps:
The enrichment of step 1, danshinolic acid Y: taking root of red-rooted salvia phenolic acid extract, is dissolved with the 1-3 times of water measured, and MCI Gel chromatographic column is added
In, the ratio of extract quality and chromatographic column filler volume is 1:60-1:20, and loading speed is 1-3BV/h, then uses 10-20%
Ethanol water elution, flow velocity 1-3BV/h are detected with HPLC method, and compareed with root of red-rooted salvia phenolic acid extract finger-print, are collected
Eluent containing danshinolic acid Y adjusts pH value 2.5-4, again with the absorption enrichment of MCI Gel chromatographic column, initial extraction amount of substance
And the ratio of chromatographic column filler volume is 1:3-1:10, and with the water elution of 3-5BV, depickling is discarded, and is finally washed with 70-95% ethyl alcohol
De-, flow velocity 1-3BV/h collects eluent, is concentrated to dryness to obtain danshinolic acid Y enriched substance；
Step 2, fine separation: danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 1-3 times of quality volume, using dynamic axial
Pressurization preparation liquid phase separation, each sample introduction mobile phase: acetonitrile -0.01% aqueous formic acid 23-25:77-75, flow velocity 5-7BV/h,
The eluent containing danshinolic acid Y is detected and merged with HPLC method, is concentrated under reduced pressure to give danshinolic acid Y monomer.
8. preparation method according to claim 7, which is characterized in that the described method comprises the following steps:
The enrichment of step 1, danshinolic acid Y: taking root of red-rooted salvia phenolic acid extract, and MCI Gel chromatography is added in the water dissolution of 2 times of quality volumes
In column, each loading extracts ratio 1:60~1:20 of object amount-MCI-Gel packing volume, flow velocity 2BV/h, uses after end of the sample
The elution of 10%~20% ethanol water, flow velocity 2BV/h adjust eluent pH value to 2.8-3.2, be added again it is regenerated with
In upper MCI Gel chromatographic column, 5BV, depickling being washed with water, eluent discards, flow velocity: 2BV/h is washed with 80-95% ethyl alcohol later
De-, flow velocity: 2BV/h collects eluent 2BV, and recycling design is concentrated to dryness to obtain danshinolic acid Y enriched substance；
Step 2, fine separation: danshinolic acid Y enriched substance is completely dissolved with the mobile phase of 2 times of quality volumes, using dynamic axial plus
Standby liquid phase separation is suppressed, each sample introduction mobile phase: -0.01% aqueous formic acid 23-25:77-75 of acetonitrile, flow velocity 6BV/h, every 1/
4BV connects a sample, and the eluent containing danshinolic acid Y is detected and merged with HPLC method, is concentrated under reduced pressure to give danshinolic acid Y monomer.
9. preparation method according to claim 1-8, which is characterized in that the step 1, wherein root of red-rooted salvia phenolic acid
Extract is the salvianolic acid extract prepared according to official method, or the Danshen injection polyphenol according to prior art preparation
The raw material of acid or the water extract of Radix Salviae Miltiorrhizae.
10. preparation method according to claim 1-8, which is characterized in that the method further includes walking
The step of rapid 2 gained danshinolic acid Y monomer is dried, the drying are 60 DEG C and are dried under reduced pressure 10-15 hours.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115068464A (en) * | 2022-06-16 | 2022-09-20 | 天津天士力之骄药业有限公司 | Application of salvianolic acid Y or pharmaceutically acceptable salt or ester thereof in preparing medicines for treating gastric polyp |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425659A (en) * | 2002-12-31 | 2003-06-25 | 南京虹桥医药技术研究所 | Process for preparing danshen salviandic acid |
| CN102351819A (en) * | 2011-10-27 | 2012-02-15 | 广州汉方现代中药研究开发有限公司 | Extraction, purification and preparation method of high-purity salvianolic acid B |
| CN105218495A (en) * | 2014-05-27 | 2016-01-06 | 天津天士力之骄药业有限公司 | A kind of red sage root water soluble ingredient new compound, preparation method and application thereof |
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2018
- 2018-01-03 CN CN201810005980.2A patent/CN109988133A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425659A (en) * | 2002-12-31 | 2003-06-25 | 南京虹桥医药技术研究所 | Process for preparing danshen salviandic acid |
| CN102351819A (en) * | 2011-10-27 | 2012-02-15 | 广州汉方现代中药研究开发有限公司 | Extraction, purification and preparation method of high-purity salvianolic acid B |
| CN105218495A (en) * | 2014-05-27 | 2016-01-06 | 天津天士力之骄药业有限公司 | A kind of red sage root water soluble ingredient new compound, preparation method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115068464A (en) * | 2022-06-16 | 2022-09-20 | 天津天士力之骄药业有限公司 | Application of salvianolic acid Y or pharmaceutically acceptable salt or ester thereof in preparing medicines for treating gastric polyp |
| CN115068464B (en) * | 2022-06-16 | 2023-03-21 | 天津天士力之骄药业有限公司 | Application of salvianolic acid Y or pharmaceutically acceptable salt or ester thereof in preparing medicines for treating gastric polyp |
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Application publication date: 20190709 |