CN110294728B - Preparation method of isolithospermic acid - Google Patents
Preparation method of isolithospermic acid Download PDFInfo
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Abstract
The invention relates to a preparation method of isolithospermic acid, namely a preparation method of 4- (2-carboxyvinyl) -2- (3, 4-dihydroxyphenyl) -2, 3-2H-7-hydroxy-3-benzofurancarboxylic acid-3- [ 1-carboxyl-2- (3, 4-dihydroxyethyl ] ester, which comprises the following steps: step 1, enrichment of isolithospermic acid, step 2: preparing an isolithospermic acid crude product, and step 3: and (3) preparing the pure isolithospermic acid. The method has the advantages of few steps, high purity and simple operation.
Description
Technical Field
The invention relates to preparation of a pharmaceutical compound, belongs to the field of pharmaceutical chemicals, and in particular relates to a preparation method of isolithospermic acid.
Technical Field
4- (2-carboxyvinyl) -2- (3, 4-dihydroxyphenyl) -2, 3-2H-7-hydroxy-3-benzofurancarboxylic acid-3- [ 1-carboxyl-2- (3, 4-dihydroxyethyl ] ester, also known as isoalkanoic acid, is an isomer of alkanoic acid, and has the structural formula I:
the isolithospermic acid is obtained by separating and reporting nuclear magnetic data of a degradation product of the salvianolic acid B aqueous solution, which is detected by utilizing liquid, from a Korean scholars after the isolithospermic acid is obtained (the related information is disclosed in the literature: yong-Xue Guo etc., kinetics and mechanism of degradation of lithospermic acid B in aqueous solution, journal of Pharmaceutical and Biomedical Analysis (2007) 1249-1255), wherein the preparation method comprises the following steps: salvianolic acid B was dissolved in water and pH was adjusted to 4.9 with HCl. The aqueous solution was then heated at 100℃for 24 hours and concentrated under reduced pressure at 39 ℃. The concentrate was purified by Sephadex LH-20 column chromatography (70-230 mesh, 2.0 x 132cm, column volume 480 ml), eluting with methanol/water (8:2, V/V). The resulting fractions were combined using thin layer chromatography or liquid phase analysis to finally obtain 23 fractions. The fractions with the volume-to-bed volume ratios of 1.30-2.40 and 3.96-4.58 are combined and purified by using a preparative liquid phase (chromatographic column: 20 x 250mm, flow rate: 9.9ml/min, detection wavelength: 280nm, mobile phase: trifluoroacetic acid adjusted pH to 2.65, acetonitrile/water 10:90V/V, elution gradient 100% A. Fwdarw.100% B,100% B for 15 min) to obtain isolithospermic acid (t) R 35.33 min). The document defines the structure of isolithospermic acid as shown in structural formula I. (see, for information on this, hylong Jae Lee etc., chemical conversions of salvianolic acid B by decoction in aqueous solution, fitolterapia 83 (2012) 1196-1204).
The process is extremely complex, uses various organic reagents, has high dependence on high-pressure separation equipment, and has long preparation period and high cost.
The salvianolic acid extract comprises a commercial salvianolic acid extract (pages 397-398 in the 1 st part of the 2015 edition of Chinese pharmacopoeia) which accords with the 2015 edition of Chinese pharmacopoeia standard and a salvianolic acid extract which is a preparation raw material of salvianolic acid for injection and is sold on the existing market (the method is various, such as a preparation method mentioned in the background technology of CN105588885A with the application number of 201410572435.3), wherein the preparation method of the commercial salvianolic acid extract which accords with the 2015 edition of Chinese pharmacopoeia standard is as follows: cutting Saviae Miltiorrhizae radix into small pieces, extracting with water at 80deg.C twice, mixing extractive solutions, filtering, concentrating the filtrate at 60deg.C under reduced pressure to obtain fluid extract with relative density of 1.18-1.22 (50deg.C), cooling, adding ethanol to ethanol content of 70%, standing for 12 hr, collecting supernatant, recovering ethanol under reduced pressure, concentrating to soft extract, and drying.
Further analysis shows that the establishment method of the fingerprint of the salvianolic acid extract is a UPLC method, and the chromatographic conditions are as follows:
instrument: waters UPLC-PDA
Chromatographic column: waters Acquity HSS T3 um 1.8
Mobile phase: a:0.1% formic acid water, B: acetonitrile
Gradient elution:
table 1: gradient elution meter
As a result, as shown in FIG. 1, the salvianolic acid extract comprises protocatechuic aldehyde, salvianolic acid D, salvianolic acid B, rosmarinic acid, salvianolic acid Y, lithospermic acid, isolithospermic acid, etc., and the total salvianolic acid extract and the raw material of salvianolic acid for injection (i.e. salvianolic acid extract) have consistent maps. Based on the above findings, the inventors have attempted to isolate isolithospermic acid from salvianolic acid extract, but: firstly, the content of the compound in the salvianolic acid extract is very low; secondly, the compound is next to salvianolic acid D in the fingerprint, which indicates that the polarities of the two compounds are very close; and the third phenolic acid compound and the phenolic acid compound both contain a plurality of phenolic hydroxyl groups, are weak acid and are easy to be oxidized and decomposed, and the separation condition is relatively harsh. Therefore, the compound is difficult to separate from the salvianolic acid extract, and the salvianolic acid D and the isolithospermic acid cannot be completely separated by adopting the prior art such as the method in the literature.
The invention aims to provide a method for separating isolithospermic acid from salvianolic acid extract, and the separation and preparation of high-purity monomer isolithospermic acid have important significance for further researching the pharmacological activity, perfecting the quality standard and improving the standard of related products.
Disclosure of Invention
The invention provides a preparation method of isolithospermic acid in red sage root, which comprises the following steps:
step 1, enrichment of isoalkanoic acid
Step 2: preparation of crude isolithospermic acid
Step 3: preparation of pure isolithospermic acid
Wherein, the enrichment of the isoalkannic acid in the step 1 comprises the following steps:
dissolving salvianolic acid extract with water, adding into reverse chromatographic column, eluting with ethanol water solution, extracting again with reverse chromatographic column solid phase, eluting with 95% ethanol, and concentrating the eluate to dry to obtain isolithospermic acid concentrate;
wherein, step 2: the preparation method of the isolithospermic acid crude product comprises the following steps:
removing pigment from the isoalkannic acid concentrate to obtain a crude isoalkannic acid concentrate;
wherein, the step 3: the preparation method of the pure isolithospermic acid comprises the following steps: further separating and purifying with ODS chromatographic column to obtain high purity isoalkannic acid monomer, and drying the obtained pure product.
Preferably, the enrichment of the isolithospermic acid in the step 1 is carried out by the following steps:
dissolving salvianolic acid extract with 1-3 times of water, adding into reverse chromatographic column, eluting with 8-15% ethanol water solution, adjusting pH to acidity, extracting with reverse chromatographic column solid phase again, eluting with 90-95% ethanol, collecting the lower extract, and concentrating to dry to obtain isoalkannic acid concentrate;
the step 2: the preparation method of the isolithospermic acid crude product comprises the following steps:
removing pigment from the isoalkannic acid concentrate to obtain a crude isoalkannic acid concentrate;
the step 3: the preparation method of the pure isolithospermic acid comprises the following steps:
the crude product is dissolved by acetonitrile, and is separated by ODS chromatographic column, the mobile phase is acetonitrile and aqueous solution containing 0.01 percent formic acid, and the volume ratio is 23:77-30:70, the flow rate is 4-8BV/h, comparing with the salvianolic acid fingerprint, collecting eluent containing isolithospermic acid, concentrating under reduced pressure, and drying.
The method comprises the following steps: wherein in step 1, the reverse chromatographic column is MCI Gel chromatographic column, polyamide chromatographic column, ODS-C18 chromatographic column or LH-20 Gel chromatographic column, preferably MCI Gel chromatographic column;
the column passing is carried out twice, the weight-volume ratio of the extract to the filler is 1:60-1:20, a step of; the second time, the weight-volume ratio of the extract to the filler is 1:6-1:3;
the twice-subsequent liquid receiving, the first column eluent is detected by an HPLC method, and simultaneously, the eluent containing isolithospermic acid is collected by comparing with the fingerprint of the salvianolic acid extract; the ethanol eluent is directly collected for the second time.
Wherein, the regulator for regulating the pH value is formic acid or hydrochloric acid; the pH value is 3.0-4.5;
the method comprises the following steps: in the step 2, the pigment removing method is LH20 gel column chromatography or active carbon decolorization, preferably LH-20 gel column chromatography; when the pigment is removed by LH20 gel column chromatography, the pigment is completely dissolved by 45-55% ethanol water solution with the mass volume of 1-3 times, and then added into LH20 gel column chromatography, and eluted by 45-55% ethanol with the flow rate of: and (3) referring to fingerprint spectrum comparison of salvianolic acid extract at 0.08-0.12BV/h, and combining crude products containing isolithospermic acid.
The method of the invention specifically comprises the following steps:
step 1, enrichment of isoalkanoic acid
Dissolving salvianolic acid extract with 1-3 times of water, and adding into reverse chromatographic column with the volume ratio of sample loading to chromatographic column filler of 1:60-1:20, loading at a loading speed of 1-3BV/h, eluting with 8-15% ethanol water solution at a flow rate of 1-3%, adjusting pH to 2.5-5 with a next liquid, and performing solid phase extraction again with a reverse chromatographic column, wherein the volume ratio of the extract to the chromatographic column filler is 1:6-1:3, eluting with 90-95% ethanol at a flow rate of 1-3BV/h, collecting eluate, and concentrating to dryness to obtain isoalkannic acid concentrate;
step 2: preparation of crude isolithospermic acid
Removing pigment from the isoalkannic acid concentrate to obtain a crude isoalkannic acid concentrate;
step 3: preparation of pure isolithospermic acid
The crude product is dissolved by acetonitrile, and the mass volume ratio of the acetonitrile to the crude product is 1-3:1, separating by ODS chromatographic column, wherein the mobile phase is acetonitrile and aqueous solution containing 0.01% formic acid, and the volume ratio is 23:77-30:70, the flow rate is 4-8BV/h, and the eluent containing salvianolic acid Y is collected by comparing with the fingerprint spectrum of isolithospermic acid, and then is concentrated and dried under reduced pressure.
More specifically, the method of the present invention may include the steps of:
step 1: enrichment of isolithospermic acid
Dissolving salvianolic acid extract in 2 times of water, and loading into MCI Gel chromatographic column (each time the extract is loaded-MCI-Gel filler 1:60-1:20), with flow rate: 2BV/h (2 times bed volume/h), eluting with 10% ethanol water solution after loading, flow rate: 2BV/h (2 bed volumes/hour), one sample per 1/10BV (1/10 bed volumes);
detecting by HPLC, and simultaneously comparing with fingerprint of salvianolic acid extract, and mixing the eluates containing isolithospermic acid. Adjusting pH of the eluate to 3.0-4.5, adding into MCI Gel chromatographic column (extract-MCI-Gel packing 1:6-1:3), washing with water for 5BV, discarding the eluate, and flowing at the following rate: 2BV/h (2 bed volumes/h), followed by elution with 95% ethanol, flow rate: 2BV/h (2 times of bed volume/h), collecting eluent 2BV, recovering solvent, concentrating to dryness to obtain isoalkannic acid concentrate;
step 2: preparation of crude isolithospermic acid
The isoalkanoic acid enrichment is completely dissolved by a 50% ethanol aqueous solution with the mass volume of 2 times, added into LH20 gel column chromatography, eluted by 50% ethanol with the flow rate: 0.1BV/h, each 500ml of eluent is connected, HPLC method is used for detection, and simultaneously, the eluent is compared with the fingerprint of salvianolic acid extract, and the crude products containing isolithospermic acid are combined;
step 3: preparation of pure isolithospermic acid crude product the crude isolithospermic acid is completely dissolved by using 2 times of acetonitrile aqueous solution with mass volume, ODS chromatographic column is separated, mobile phase: acetonitrile-aqueous solution containing 0.01% formic acid, the volume ratio is 23:77-30:70 (), the flow rate is 6BV/h, every 1/4BV is connected with one sample, HPLC method is used for detection, meanwhile, the eluent containing isolithospermic acid is combined with the fingerprint of salvianolic acid, the isolithospermic acid monomer is obtained by decompression concentration and drying, and the reduced pressure drying is carried out at 60 ℃ for 12 hours.
The preparation method of the invention has the following advantages:
1. the inventors found that the salvianolic acid extract contains a small amount of isolithospermic acid, and made a large number of experiments to obtain the compound from the salvianolic acid extract.
The salvianolic acid compound is mostly separated by a silica gel column in a rough way, and then is separated by a reversed-phase C18 column repeatedly. The various organic reagents used have high dependence on high pressure separation equipment.
The conventional process of phenolic acid compounds is used for reference in the development process of the preparation process, such as comparative example 1, the compound isolithospermic acid is separated by a silica gel column firstly, then separated by an ODS (C18 filler) chromatographic column, and finally only the mixture of the compound isolithospermic acid and the salvianolic acid D can be obtained (the HPLC detection spectrum of the mixture is shown in figure 2). The method has long separation period, uses a large amount of organic solvent, and brings hidden danger to operators.
In order to separate the compound from the salvianolic acid D, an experimenter screens different types of ODS columns, the purpose is not achieved, and finally, the original point is returned to a rescreening method, and finally, the method of separating the compound from the salvianolic acid D by gel column chromatography and purifying the ODS is determined.
2. In the prior art, macroporous adsorption resin columns, ODS reverse chromatography and repeated HPLC are adopted to separate phenolic acid compounds, but the inventor finds that when the method is used for referencing the method to separate isolithospermic acid and salvianolic acid D from the salvianolic acid extract, the salvianolic acid D is extremely easy to be converted into another unknown compound and always mixed into the fraction of the isolithospermic acid when the method is adopted to separate the same raw materials, such as the ODS columns or the HPLC, so that single isolithospermic acid cannot be obtained.
3. Separating the isoalkannic acid concentrate by LH20 gel column chromatography to obtain crude isoalkannic acid, and purifying by C18 reversed phase chromatographic column to obtain high-purity isoalkannic acid with content of more than 98.5%. The method has few steps, high purity and simple operation, and can be used for developing new drugs of cardiovascular and cerebrovascular systems.
Drawings
Fig. 1: fingerprint spectrum of salvianolic acid extract, and specific establishment method is described in background art;
fig. 2: HPLC detection profile of isolithospermic acid and salvianolic acid D mixture;
fig. 3: an enriched isolithospermic acid UPLC detection diagram;
fig. 4: UPLC detection chart of isolithospermic acid crude product
Fig. 5: and (3) an isolithospermic acid monomer UPLC detection diagram.
4. Detailed description of the preferred embodiments
The medicaments of the present invention are further illustrated by the following examples, which are given by way of illustration only and are not intended to be limiting.
Example 1: preparation method of isolithospermic acid
Step one, enrichment of isolithospermic acid
Taking 500g of salvianolic acid extract, dissolving with 1000ml of water, loading 100ml each time, and adding into a glass chromatographic column filled with 3L of MCI Gel at a flow rate: 100ml/min. Eluting with 10% ethanol water solution after loading, and flowing speed: 100ml/min, one sample per 300ml from the color of the following stock. And (3) collecting 25 bottles of sample liquid, detecting by using a UPLC method, simultaneously comparing with the salvianolic acid fingerprint, and combining 2-6 bottles of eluent containing isolithospermic acid. The pH value of the eluent is adjusted to 3.0, the eluent is added into a glass chromatographic column filled with 3L of MCI Gel again, 15L of the eluent is washed by water, the eluent is discarded, and the flow rate is that: 100ml/min. Then eluting with 95% ethanol, flow rate: 100ml/min, collecting eluent 6L, recovering solvent, concentrating to dryness to obtain isoalkannic acid concentrate. The above steps were performed in parallel to complete the separation of all samples, giving 45g of concentrate, lot number 20150530.
Step two: preparation of isolithospermic acid enriched crude product
45g of isoalkanoic acid concentrate is completely dissolved in 90ml of 50% aqueous ethanol, and the mixture is put into a glass column filled with 1kg of LH-20 sephadex, eluted with 50% ethanol, flow rate: 10ml/min, each 500ml was followed by one eluent. Detecting by UPLC method, and simultaneously comparing with salvianolic acid fingerprint, combining 12.3g crude product containing isolithospermic acid, and lot number 20150609.
Step three: preparation of pure product of isoalkannic acid enrichment
12.3g of isolithospermic acid crude product is completely dissolved by 25ml of 23% acetonitrile aqueous solution, liquid phase separation is prepared by dynamic axial pressurization, 10ml of sample injection is carried out each time, and the mobile phase is as follows: acetonitrile-water 23:77 (0.01% formic acid), flow rate 200ml/min, one sample per 500 ml. Detecting by UPLC method, and simultaneously comparing with salvianolic acid fingerprint, and mixing eluates containing isolithospermic acid concentrate. Concentrating under reduced pressure to obtain pure isolithospermic acid, separating all samples by the above operation, and drying at 60deg.C under reduced pressure for 12 hr. Product lot 20150622.
Nuclear magnetic resonance testing was performed on the resulting product, and the resulting nuclear magnetic data (see table 2) were consistent with literature reports (hyuang Jae Lee etc., chemical conversions of salvianolic acid B by decoction in aqueous solution, fitterapia 83 (2012) 1196-1204), demonstrating that the resulting compound was isolithospermic acid.
Table 2: isolithospermic acid nuclear magnetic resonance data attribution
Experimental example 1: content determination of Isonoalkannic acid
1. Test method
Chromatographic conditions
Chromatographic column: waters Aqucity UPLCTM HSS T3 (2.1X105 mm,1.8 μm)
Chromatographic conditions: λ=280 nm; column temperature: 40 ℃; flow rate: 0.4ml/min; mobile phase: gradient elution was used and is shown in Table 3
Table 3 gradient table for determining mobile phase of phenolic acid standard in red sage root
(II) preparation of sample solution to be tested
30mg of isolithospermic acid concentrate 20150530 (prepared by the method of step 1 in example 1) was weighed, placed in a 25ml volumetric flask, dissolved with methanol, and fixed to a scale for measurement.
20mg of isolithospermic acid crude product 20150509 (prepared by the method of step 2 in example 1) is weighed, placed in a 25ml volumetric flask, dissolved by methanol, and fixed to a scale for measurement.
10mg of high-purity isolithospermic acid 20150620 (prepared by the method of step 1 in example 1) is taken and placed in a 25ml volumetric flask, dissolved by methanol, and fixed to a scale for measurement.
(3, measurement)
And respectively taking 2 mu L of each sample solution, measuring, and recording the percentage content of the isolithospermic acid by a normalization method.
4. The content measurement results are shown in Table 4, FIG. 3 and FIG. 4.
Table 4: isolithospermic acid sample content determination result
| Sample name | Percentage content (%) | Remarks |
| Isolithospermic acid concentrate 20150530 | 42.3％ | See FIG. 3 |
| Crude isolithospermic acid 20150509 | 92.6％ | See FIG. 5 |
| High purity isoalkanoic acid 20150620 | 98.7％ | See FIG. 4 |
The results show that: the isolithospermic acid obtained by the method has high purity.
Comparative example 1: separation and detection
1. Separation method
Step 1, silica gel column chromatography separation of isoalkannic acid
Dissolving 50g of salvianolic acid extract in 200ml of water, extracting with equal volume of ethyl acetate for 3 times, collecting ethyl acetate extract, recovering solvent under reduced pressure, adding 80g of 100-200 mesh silica gel, and stirring.
Filling a silica gel column: 1000g of raw material 200-300 mesh silica gel is taken and ethyl acetate-petroleum ether 1:1 (2% formic acid) for 30min, packing with column, and using ethyl acetate-petroleum ether 1:1 balance 3BV.
Separating: the samples were dry loaded with ethyl acetate-petroleum ether 1:1 (2% formic acid), about 0.2BV as unit, and TLC detection, and development with 5% sulfuric acid ethanol as developer.
Fraction combination: and (3) combining fractions consistent with the spot Rf value of the isolithospermic acid reference substance, and concentrating under reduced pressure to dryness to obtain 15g of crude product containing isolithospermic acid.
Step 2, fine separation
15g of crude isolithospermic acid is completely dissolved by 30ml of 23% acetonitrile aqueous solution, the liquid phase (filled with reversed phase C18 filler) is prepared by dynamic axial pressurization and separated, 10ml of liquid phase is injected each time, and the mobile phase: acetonitrile-water 23:77 (0.01% formic acid), flow rate 200ml/min, one sample per 500 ml. Detecting by HPLC, and simultaneously combining the eluates containing the enriched matters with the fingerprint of salvianolic acid. The mixture was concentrated under reduced pressure to give isolithospermic acid, and all samples were separated in parallel to give 0.8g of lot number 20150305.
2. Detection method
The product is detected by the following steps:
experimental instrument and materials:
2.1Agilent1100 high performance liquid chromatograph
2.2HPLC detection conditions:
2.3 chromatography column: agilent ZORBAX SB-C18, 5um, 4.6 x 250mm, sample injection amount 10ul, flow rate 1ml/min, detection wavelength 280nm, column temperature 30 ℃. Mobile phase a was 0.02% phosphoric acid water and B was 80% acetonitrile-20% 0.02% phosphoric acid water. Elution gradient 0min,90% a;8min,78% A;15min,74% A;35min,61% A;40min,90% A;50min,90% A.
3. The results are shown in FIG. 2
The results show that: the use of the reversed-phase C18 filler does not separate isolithospermic acid from salvianolic acid D, and an unknown compound is easily present during concentration after separation.
Claims (9)
1. The preparation method of isolithospermic acid is characterized by comprising the following steps:
step 1: enrichment of isolithospermic acid; dissolving salvianolic acid extract with water, adding into reverse chromatographic column, eluting with ethanol water solution, extracting again with reverse chromatographic column solid phase, eluting with 95% ethanol, and concentrating the eluate to dry to obtain isolithospermic acid concentrate;
step 2: preparing an isolithospermic acid crude product; removing pigment from the isoalkannic acid concentrate to obtain crude isoalkannic acid;
step 3: preparing pure isolithospermic acid; further separating and purifying with ODS chromatographic column to obtain high purity isoalkannic acid monomer, and drying the obtained pure product.
2. The method of claim 1, wherein the enrichment of isoalkanoic acid in step 1 is as follows:
dissolving salvianolic acid extract with 1-3 times of water, adding into reverse chromatographic column, eluting with 8-15% ethanol water solution, adjusting pH to acidity with the next liquid, extracting with reverse chromatographic column solid phase again, eluting with 95% ethanol, collecting the next liquid, and concentrating to dry to obtain isolithospermic acid concentrate.
3. The method of claim 1, wherein step 3: the preparation method of the pure isolithospermic acid comprises the following steps:
the crude product is dissolved by acetonitrile, and is separated by ODS chromatographic column, the mobile phase is acetonitrile and aqueous solution containing 0.01 percent formic acid, and the volume ratio is 23:77-30:70, comparing the flow rate with 4-8BV/h with the fingerprint of salvianolic acid extract, collecting eluent containing isolithospermic acid, concentrating under reduced pressure, and drying.
4. The method according to claim 2, wherein,
wherein in the step 1, the reverse chromatographic column is an MCI Gel chromatographic column, a polyamide chromatographic column, an ODS-C18 chromatographic column or an LH-20 Gel chromatographic column;
wherein, the extract is added into the reverse chromatographic column for the first time, and the weight volume ratio of the extract to the filler is 1:60-1:20, a step of; extracting with reverse chromatographic column at a weight/volume ratio of 1:6-1:3;
the lower liquid is detected by HPLC, and the eluent containing isolithospermic acid is collected by comparing with the fingerprint of salvianolic acid extract; the ethanol eluent is directly collected for the second time,
wherein, the regulator for regulating the pH value is formic acid or hydrochloric acid; the pH value is 3.0-4.5.
5. The method according to claim 4, wherein in step 1, the reverse chromatography column is an MCI Gel chromatography column.
6. The method according to claim 1, wherein,
in the step 2, the pigment removing method is LH20 gel column chromatography or active carbon decolorization.
7. The method according to claim 6, wherein in step 2, the method of removing the pigment is LH-20 gel column chromatography; when the pigment is removed by LH20 gel column chromatography, the pigment is completely dissolved by 45-55% ethanol water solution with the mass volume of 1-3 times, and then added into LH20 gel column chromatography, and eluted by 45-55% ethanol with the flow rate of: and (3) referring to fingerprint spectrum comparison of salvianolic acid extract at 0.08-0.12BV/h, and combining crude products containing isolithospermic acid.
8. The method of manufacturing according to claim 1, comprising the steps of:
step 1, enrichment of isoalkanoic acid
Dissolving salvianolic acid extract with 1-3 times of water, and adding into reverse chromatographic column with the volume ratio of sample loading to chromatographic column filler of 1:60-1:20, the sample loading speed is 1-3BV/h, then the sample is eluted by 8-15% ethanol water solution, the flow rate is 1-3%, the pH value is adjusted to 2.5-5 by liquid connection, the solid phase extraction is carried out again by a reverse chromatographic column, and the volume ratio of the extract to the chromatographic column filling is 1:6-1:3, eluting with 95% ethanol at a flow rate of 1-3BV/h, collecting eluate, and concentrating to dryness to obtain isolithospermic acid concentrate;
step 2: preparation of crude isolithospermic acid
Removing pigment from the isoalkannic acid concentrate to obtain a crude isoalkannic acid concentrate;
step 3: preparation of pure isolithospermic acid
The crude product is dissolved by acetonitrile, and the mass volume ratio of the acetonitrile to the crude product is 1-3:1, separating by ODS chromatographic column, wherein the mobile phase is acetonitrile and aqueous solution containing 0.01% formic acid, and the volume ratio is 23:77-30:70, the flow rate is 4-8BV/h, comparing with the fingerprint of salvianolic acid extract, collecting eluent containing isolithospermic acid, concentrating under reduced pressure, and drying.
9. The method of manufacturing according to claim 1, comprising the steps of:
step 1: enrichment of isolithospermic acid
Dissolving salvianolic acid extract in 2 times of water, adding into MCI Gel chromatographic column, and loading the extract amount-MCI-Gel filler 1:60-1:20 at each time at the flow rate: 2BV/h, eluting with 10% ethanol water solution after loading, flow rate: 2BV/h, one sample per 1/10 BV;
detecting by HPLC, simultaneously comparing with fingerprint of salvianolic acid extract, mixing eluates containing isolithospermic acid, adjusting pH to 3.0-4.5, adding into MCI Gel chromatographic column again, washing with water 5: 5BV, discarding the eluates, and flowing at the rate of 1:6-1:3: 2BV/h, followed by elution with 95% ethanol, flow rate: 2BV/h, collecting eluent 2BV, recovering solvent, concentrating to dryness to obtain isoalkannic acid concentrate;
step 2: preparation of crude isolithospermic acid
The isoalkanoic acid enrichment is completely dissolved by a 50% ethanol aqueous solution with the mass volume of 2 times, added into LH20 gel column chromatography, eluted by 50% ethanol with the flow rate: 0.1BV/h, each 500ml of eluent is connected, HPLC method is used for detection, and simultaneously, the eluent is compared with the fingerprint of salvianolic acid extract, and the crude products containing isolithospermic acid are combined;
step 3: preparation of pure isolithospermic acid
The isolithospermic acid crude product is completely dissolved by acetonitrile water solution with the mass volume of 2 times, ODS chromatographic column is separated, and the mobile phase is: acetonitrile-aqueous solution containing 0.01% formic acid, the volume ratio is 23:77-30:70, the flow rate is 6BV/h, every 1/4BV is connected with one sample, the detection is carried out by an HPLC method, meanwhile, the detection is compared with the fingerprint of the salvianolic acid extract, the eluent containing isolithospermic acid is combined, the isolithospermic acid monomer is obtained by decompression concentration and drying, and the decompression drying is carried out at 60 ℃ for 12 hours.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317000A (en) * | 2015-07-06 | 2017-01-11 | 天津天士力之骄药业有限公司 | Salvianolic acid compound as well as preparation method and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106317000A (en) * | 2015-07-06 | 2017-01-11 | 天津天士力之骄药业有限公司 | Salvianolic acid compound as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Biotransformation of Salvianolic acid B by Fusarium oxysporum f. sp. Cucumerinum and Its Two Degradation Routes;Shidong Kan et al.;《Natural Product Communications》;20121231;第7卷(第7期);第885-888页 * |
| 丹酚酸B的降解机理及纯化工艺研究;郭永学;《中国博士学位论文全文数据库 工程科技Ⅰ辑》;20080515(第5期);第B016-14页 * |
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