CN111454319B - Method for separating and preparing trace components from sodium aescinate - Google Patents
Method for separating and preparing trace components from sodium aescinate Download PDFInfo
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- CN111454319B CN111454319B CN202010283301.5A CN202010283301A CN111454319B CN 111454319 B CN111454319 B CN 111454319B CN 202010283301 A CN202010283301 A CN 202010283301A CN 111454319 B CN111454319 B CN 111454319B
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Abstract
The invention discloses a method for separating and preparing a trace component from sodium aescinate, which comprises the following two steps: 1) loading sodium aescinate raw material medicine to a macroporous resin column, eluting with ethanol, collecting eluent, concentrating, drying, then loading to a silica gel column for chromatography, eluting with a mixed solvent of chloroform and methanol, collecting eluent containing target components, and concentrating to dryness to obtain a crude product; 2) the crude product is continuously subjected to two-time preparative liquid chromatography purification, the target fraction is collected, concentrated and dried, the component monomer of the compound is effectively obtained by adopting the preparation method, and a material basis is provided for the secondary development of sodium aescinate and the research of effect substances. The purity of the monomer compound prepared by the invention is up to more than 95%, the product yield is about 1.5%, and the method has good industrial production prospect.
Description
Technical Field
The invention relates to a method for separating and preparing a trace component from aescine sodium raw material medicines, belonging to the field of pharmacy.
Background
Aescin sodium is a natural medicine, is total saponin sodium salt extracted from buckeye seed of aesculus plant of aesculaceae, has the functions of resisting cerebral edema and resisting inflammation clinically, and is mainly used for treating chronic venous insufficiency. The aescin sodium has its main active components named as aescin A, B, C, D through liquid chromatographic analysis and has its peaks named as shown in figure 1, but the aescin sodium also contains other trace components.
Under the heat tide of the modernization of natural plant drugs, based on the complex diversity of components, the other components except for the aescin A, B, C, D are particularly important in the secondary development of natural drugs, and many new substances with remarkable biological activity are not lacked. In addition, the explanation of the effect substance basis of the sodium aescinate and the improvement of the drug quality standard are also important preconditions for the international certification of natural plant drugs, which requires the effective preparation and separation of component monomers in the sodium aescinate to obtain a large amount of monomer components, thereby providing a substance basis for the subsequent quality and pharmacological toxicological studies.
At present, according to the HPLC chromatographic method for detecting sodium aescinate provided in Chinese pharmacopoeia, only the raw material medicines are quantitatively and qualitatively analyzed on an analytical column, the sample volume is very small, and the raw material medicines cannot be amplified to a preparation column for monomer preparation. Therefore, most documents such as CN102020691, CN102532242 report methods for preparing monomer of aescin A, B, C, D, and there is no effective preparation method for preparing trace components with small content in aescin sodium at present, especially for components with poor separation degree under conventional HPLC analysis and detection, which is more difficult.
Disclosure of Invention
The invention aims to provide a method for separating and preparing a trace component from sodium aescinate.
A method for separating and preparing a trace component from sodium aescinate, wherein the chemical structural formula of the trace component is as follows:
the method comprises the following two steps:
1) loading sodium aescinate raw material medicine to a macroporous resin column, eluting with ethanol, collecting eluent, concentrating, drying, then loading to a silica gel column for chromatography, eluting with a mixed solvent of chloroform and methanol, collecting eluent, and concentrating to dryness to obtain a crude product;
2) and continuously carrying out two times of preparative liquid chromatography purification on the crude product, collecting target fractions, concentrating and drying.
Preferably, the macroporous resin is CHP20P type.
Preferably, the ethanol elution comprises two steps of firstly eluting with 30-45% ethanol, then eluting with 50-70% ethanol, and collecting 50-70% ethanol eluate.
Preferably, the volume ratio of chloroform to methanol is 1-5: 1.
preferably, the chromatographic columns used for the two preparative liquid chromatography purifications are reverse phase chromatographic columns, the packing materials of the chromatographic columns are polystyrene/divinylbenzene polymers, and the volume ratio of the mobile phase is 60-70: the mixed solution of 40-30% methanol and 0.5-2% formic acid is eluted at equal rate, and the detection wavelength is 200-.
Preferably, the filler used in the first purification has a particle size of 10-15 μm and a pore size of 300-500A, and the filler used in the second purification has a particle size of 3-5 μm and a pore size of 100-300A.
Preferably, the elution time of the first purification is 100-150 min, the flow rate is 20-50 mL/min, the elution time of the second purification is 70-90 min, and the flow rate is 5-15 mL/min.
In the invention, formic acid, ethanol and methanol have volume concentration, and the solvent is water, for example, 1 per thousand formic acid means that 1ml of formic acid is contained in 1000ml of formic acid aqueous solution.
The invention has the beneficial effects that:
the chromatographic peak time of the compound prepared by the invention in conventional HPLC analysis and detection is about 136min (figure 1), two substances with very similar chemical structures and physical and chemical properties interfere in front and at back, and the compound is difficult to separate by adopting conventional liquid chromatography. The purity of the monomer compound prepared by the invention is up to more than 95%, the product yield is about 1.5%, and the method has good industrial production prospect.
Drawings
FIG. 1 is a liquid chromatography analysis location chart of sodium aescinate bulk drug.
FIG. 2 is a separation chromatogram of one reverse preparation in example 1.
FIG. 3 is a separation chromatogram of the secondary reverse preparation in example 1.
Fig. 4 is an HMBC correlation spectrum.
Detailed Description
The following examples further illustrate the invention but do not limit it accordingly. The aescine sodium raw material used in the following examples was produced by wuhan aimin pharmaceutical products limited company, and the purity thereof was 99.7%.
Example 1
1. Preparation of crude product
Taking 1L of CHP20P macroporous resin (Mitsubishi chemical), loading into a column by a wet method, weighing 5g of sodium aescinate powder, ultrasonically dissolving the powder with 10% ethanol, eluting the solution with 45% ethanol for 5 times of the column volume, discarding, eluting with 70% ethanol for 8 times of the column volume, collecting 70% ethanol eluate, and concentrating under reduced pressure to dry to obtain 721mg dry extract. Dissolving the dry extract with 10ml of methanol by ultrasound, mixing with 10g of 80-100 mesh silica gel, loading onto a silica gel chromatographic column (the proportion of the dry extract on the column: silica gel is 1:150, w/w), eluting with chloroform/methanol (5:1, v/v) mixed solvent as eluent, collecting the eluent containing the target component, and concentrating to dryness to obtain 402mg of crude product.
2. Preparation in one step
402mg of the crude product obtained in step 1 was dissolved in 50ml of 65% methanol, and the solution was subjected to primary preparation after filtration through a 0.45 μm microporous membrane. A prepared liquid chromatography system is adopted, a column model DAC50, a filler Unips10 x 300, a particle size of 10 mu m, a pore diameter of 300A, a mobile phase of methanol/1 per mill formic acid (66: 34, v/v), a detection wavelength of 200nm, an elution time of 110min, a flow rate of 35mL/min, a sample loading amount of 5mL, and continuous trocar sampling is adopted.
Receiving component peak flow components with different retention time in segments, performing high performance liquid analysis by rotary dry inspection, and retaining flow components with peak appearance time consistent with target component peak appearance time in bulk drug liquid chromatography positioning chart (figure 1), wherein the peak appearance time of the flow components in a primary preparation separation chromatogram chart (figure 2) is 88-91 min.
And (3) carrying out reduced pressure concentration and drying on the fraction with the peak-appearing time period of 88-91min to obtain 152mg of enriched sample powder of the target compound, carrying out high performance liquid analysis on the enriched sample powder to detect the purity, and processing the analysis result by adopting a peak area normalization method to obtain the purity of 83%.
3. Second preparation
The obtained 152mg enriched sample powder was sonicated with 65% methanol until completely dissolved at a concentration of 30mg/mL, and filtered through a 0.45 μm microporous membrane for secondary preparation. The prepared liquid chromatographic column filler is Unips5 multiplied by 100, the particle size is 5 mu m, the pore diameter is 100A, the mobile phase is methanol/1 per mill formic acid (70: 30, v/v), the detection wavelength is 210nm, the elution time is 85min, the flow rate is 10mL/min, the sample injection amount is 0.5mL, and the sample injection is carried out through a continuous trocar.
Receiving component peak flow fractions with different retention time in segments, performing high performance liquid analysis by rotary dry inspection, retaining flow fractions with peak appearance time consistent with target component peak appearance time in bulk drug liquid chromatography positioning chart (figure 1), and peak appearance time segment of the flow fractions in secondary preparation separation chromatogram chart (figure 3) is 68-74 min.
And (3) carrying out reduced pressure concentration and drying on the fraction with the peak-out time period of 68-74min to obtain a solid powder sample of 92mg, carrying out high performance liquid analysis on the final product to detect the purity, and processing the analysis result by adopting a peak area normalization method to obtain the purity of 97.2%.
Wherein, the high performance liquid analysis and detection conditions of the raw material medicine, the elution fraction and the product are as follows: a chromatographic column: c18 column, 5 μm, 4.6X 250 mm; the detection wavelength is 200 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; sample introduction volume: 20 mu L of the solution; the mobile phase system is acetonitrile-1 ‰ formic acid; the elution gradient program was:
| time min | Phase A% (acetonitrile) | B phase% (1 ‰ formic acid) |
| 0 | 25 | 75 |
| 20 | 33 | 67 |
| 32 | 33 | 67 |
| 33 | 34 | 66 |
| 110 | 34 | 66 |
| 150 | 60 | 40 |
| 160 | 25 | 75 |
| 170 | 25 | 75 |
Example 2
1. Preparation of crude product
Taking 1L of CHP20P macroporous resin (Mitsubishi chemical), packing into column by wet method, weighing 5g of sodium aescinate powder, ultrasonically dissolving with 10% ethanol, loading the solution onto column, eluting with 35% ethanol for 6 times of column volume, discarding, eluting with 55% ethanol for 5 times of column volume, collecting 55% ethanol eluate, and concentrating under reduced pressure to dry to obtain 887mg dry extract. Dissolving the dry extract with 20ml methanol under ultrasound, mixing with 20g 80-100 mesh silica gel, loading onto silica gel column (the ratio of dry extract on column: silica gel is 1:150, w/w), performing column chromatography with chloroform/methanol (2:1, v/v) mixed solvent as eluent, collecting eluate containing target components, and concentrating to dry to obtain 459mg crude product.
2. Preparation in one step
Taking 459mg of the crude product in the step 1, dissolving with 50ml of 65 percent methanol, and filtering with a 0.45 mu m microporous membrane to prepare the product once. A liquid chromatography system is prepared, wherein the column model is DAC50, the filler is Unips10 x 300, the particle size is 15 mu m, the pore diameter is 500A, the mobile phase is methanol/0.5 per mill formic acid (62: 38, v/v), the detection wavelength is 200nm, the elution time is 135min, the flow rate is 25mL/min, the sample loading amount is 5mL, and the sample is injected through a continuous trocar.
Receiving component peak flow components with different retention time in segments, performing high performance liquid chromatography by rotary dry inspection, and retaining flow components with peak appearance time consistent with target component in bulk drug liquid chromatography positioning chart (figure 1). And concentrating the fraction under reduced pressure and drying to obtain 177mg of enriched sample powder of the target compound, carrying out high performance liquid analysis on the enriched sample powder to detect the purity, and processing the analysis result by adopting a peak area normalization method to obtain the purity of 77%.
3. Second preparation
177mg of the obtained enriched sample powder was sonicated with 65% methanol until completely dissolved at a concentration of 20mg/mL, and filtered through a 0.45 μm microporous membrane for secondary preparation. The chromatographic column packing is Unips5 × 100, the particle size is 5 μm, the pore diameter is 300A, the mobile phase is methanol/0.5 ‰ formic acid (65: 35, v/v), the detection wavelength is 210nm, the elution time is 88min, the flow rate is 5mL/min, the sample injection amount is 0.5mL, and the sample injection is carried out through a continuous trocar.
Receiving component peak flow components with different retention time in segments, performing high performance liquid chromatography by rotary dry inspection, and retaining flow components with peak appearance time consistent with target component in bulk drug liquid chromatography positioning chart (figure 1). And concentrating the fraction under reduced pressure, drying to obtain a solid powder sample of 98mg, performing high performance liquid analysis on the final product to detect the purity, and processing the analysis result by adopting a peak area normalization method to obtain the purity of 95.8%.
Example 3
1. Preparation of crude product
Taking 1L of CHP20P macroporous resin (Mitsubishi chemical), packing into column by wet method, weighing 5g of sodium aescinate powder, ultrasonically dissolving with 30% ethanol, loading the solution onto column, eluting with 30% ethanol for 8 times of column volume, discarding, eluting with 65% ethanol for 6 times of column volume, collecting 65% ethanol eluate, and concentrating under reduced pressure to dry to obtain 705mg dry extract. Dissolving the dry extract with 10ml of methanol by ultrasound, mixing with 10g of 100-200-mesh silica gel, loading on a silica gel chromatographic column (the proportion of the dry extract on the column: silica gel is 1:100, w/w), then performing column chromatography by using a chloroform/methanol (1:1, v/v) mixed solvent as an eluent, collecting eluent containing target components, and concentrating until the eluent is dried to obtain 386mg of crude product.
2. Preparation in one step
386mg of the crude product obtained in the step 1 is taken, dissolved by 50ml of 65 percent methanol and filtered by a 0.45 mu m microporous membrane to prepare the product for one time. A prepared liquid chromatography system is adopted, a column model DAC50, a filler Unips10 x 300, a particle size of 10 mu m, a pore diameter of 500A, a mobile phase of methanol/2 per mill formic acid (69: 31, v/v), a detection wavelength of 200nm, an elution time of 105min, a flow rate of 50mL/min, a sample loading amount of 5mL, and continuous trocar sampling is adopted.
Receiving component peak flow components with different retention time in segments, performing high performance liquid chromatography by rotary dry inspection, and retaining flow components with peak appearance time consistent with target component in bulk drug liquid chromatography positioning chart (figure 1). And concentrating the fraction under reduced pressure and drying to obtain 171mg of enriched sample powder of the target compound, carrying out high performance liquid analysis on the enriched sample powder to detect the purity, and processing the analysis result by adopting a peak area normalization method to obtain the purity of 73%.
3. Second preparation
171mg of the obtained enriched sample powder was sonicated with 65% methanol until completely dissolved at a concentration of 20mg/mL, and filtered through a 0.45 μm microfiltration membrane for secondary preparation. The chromatographic column filler is Unips5 × 100, the particle size is 3 μm, the pore diameter is 100A, the mobile phase is methanol/2 ‰ formic acid (60: 40, v/v), the detection wavelength is 210nm, the elution time is 73min, the flow rate is 15mL/min, the sample injection amount is 0.5mL, and the sample injection is carried out through a trocar continuously.
Receiving component peak flow components with different retention time in segments, performing high performance liquid chromatography by rotary dry inspection, and retaining flow components with peak appearance time consistent with target component in bulk drug liquid chromatography positioning chart (figure 1). Concentrating the fraction under reduced pressure, drying to obtain solid powder sample 85mg, performing high performance liquid analysis on the final product to detect purity, and processing the analysis result by peak area normalization to obtain purity of 95.1%.
EXAMPLE 4 confirmation of Compound Structure
A compound: white powder, molecular formula by mass spectrometry: ESI-MS (neg.): 1169[ M-H ]]-,HRESI-MS(neg.):1169.5745(C58H89O24Calcd.1169.5749) is determined as C58H90O24. Aglycone part and sugar chain part of compound1H、13C-NMR (see tables 1 and 2) data are almost identical to spectral data reported in the literature (Z.Z.Zhang Chem Pharm Bull, 1999, 47(11), 1515-. The compound contains a group of angeloyl (angeloyl) signals [ delta ]H:5.90(1H,q,J＝7.2Hz),2.04(3H,d,J＝7.2Hz),1.98(3H,s)；δC:167.9,129.2,136.6,15.8,21.0]A group of crotonyl (tigloyl) signals [ delta ]H:6.93(1H,q,J＝7.2Hz),1.43(3H,d,J＝7.2Hz),1.82(3H,s)；δC:168.3,129.2,137.2,14.1,12.3]. By careful analysis of the HMBC-related signal of the compound it was found that: h-21[ delta ]H6.74(1H,d,J＝10.1Hz)]With angeloyl (angeloyl)Ester carbonyl Signal of (C-1' (delta)C167.9) has a strong correlation, it can be determined that angeloyl (angeloyl) is linked at the C-21 position; h-22[ delta ]H 6.28(1H,d,J＝10.1Hz)]The ester carbonyl signal with crotonyl (tigloyl) C-1' (deltaC168.3) is strongly correlated, it can be determined that crotonyl (tigloyl) is attached at the C-22 position. The detailed HMBC-related signals are shown in fig. 4.
1H、13The C-NMR spectrum shows a signal having three sugar end groups, deltaH 4.90(1H,d,J＝7.7Hz,GlcA-1′)/δC104.6,δH 5.62(1H,d,J＝7.7Hz,Glc-1″)/δC104.3, and δH 5.21(1H,d,J＝7.8Hz,Glc-1″′)/δC104.8, the sugar chain moiety was shown to be a triglycoside, and all of them were presumed to be β -type sugars from the coupling constant.13One carboxycarbonyl signal (. delta.) in C-NMR spectraC172.5) indicates the presence of a glucuronic acid. The corresponding terminal hydrogen delta of the uronic acidH4.90(1H, d, J ═ 7.7Hz) with the aglycon parent C-3 (. delta.) (II)C91.1) the presence of HMBC indicates that glucuronic acid still forms a glycosidic bond with the 3-position of the parent nucleus. Another 2 glycosyl-terminal hydrogens in HMBC spectrumH5.62(1H, d, J ═ 7.7Hz, Glc-1 "), 5.21(1H, d, J ═ 7.8Hz, Glc-1 '"), respectively, and GlcA C-2 ' (δ -1 ' ") separatelyC79.7) and C-4' (delta)C82.1) is related, indicating that the sugar chain is still 3-O- [ beta-D-glucosyl (1 → 2) - [ beta-D-glucosyl (1 → 4)]- β -D-glucuronyl.
In conclusion, the compound was identified as 21 β -O-angeloyl-22 α -O-crotonyl orthoescin-3 β -O- [ β -D-glucosyl group (1 → 2) - [ β -D-glucosyl group (1 → 4) ] - β -D-glucuronide by performing nuclear magnetic signal analysis on the compound in combination with literature.
Of the compounds of Table 11H spectral data (. delta.)H 8.71ppm)
Of the compounds of Table 213C spectral data (. delta.)C 149.9ppm)
| No. | δC |
| 3 | 91.1 |
| 16 | 68.5 |
| 21 | 78.7 |
| 22 | 74.0 |
| 24 | 63.3 |
| 28 | 63.4 |
| 1′ | 104.6 |
| 2′ | 79.7 |
| 3′ | 76.6 |
| 4′ | 82.1 |
| 5′ | 76.0 |
| 6′ | 172.5 |
| 1″ | 104.3 |
| 2″ | 75.8 |
| 3″ | 78.1 |
| 4″ | 69.7 |
| 5″ | 78.3 |
| 6″ | 61.6 |
| 1″′ | 104.8 |
| 2″′ | 75.0 |
| 3″′ | 78.1 |
| 4″′ | 71.5 |
| 5″′ | 78.5 |
| 6″′ | 62.4 |
| 1″″ | 167.9 |
| 2″″ | 129.2 |
| 3″″ | 136.6 |
| 4″″ | 15.8 |
| 5″″ | 21.0 |
| 1″″′ | 168.3 |
| 2″″′ | 129.2 |
| 3″″′ | 137.2 |
| 4″″′ | 14.1 |
| 5″″′ | 12.3 |
Claims (1)
1. A method for separating and preparing a trace component from sodium aescinate is characterized by comprising the following steps: the micro-components have the following structural formula
1) preparation of crude product
Loading sodium aescinate raw material medicine to a CHP20P type macroporous resin column, eluting with 45% ethanol, then eluting with 70% ethanol, collecting 70% ethanol eluent, concentrating, drying, then loading to a silica gel column for chromatography, and performing chromatography by using a volume ratio of 5:1, eluting with a chloroform-methanol mixed solvent, collecting the eluent containing the target component, and concentrating to dryness to obtain a crude product;
2) preparation in one step
Dissolving the crude product obtained in the step 1) by using 65% methanol, filtering the solution by using a 0.45-micron microporous filter membrane, and then carrying out one-time preparation, wherein a preparation liquid chromatography system is adopted, the column type number is DAC50, the particle size of a filler is 10 microns, the pore diameter is 300A, and the volume ratio of a mobile phase is 66: 34 of methanol/1 ‰ formic acid, the detection wavelength is 200nm, the elution time is 110min, the flow rate is 35mL/min, the sample loading amount is 5mL, the sample is continuously injected through a trocar,
receiving component peak fractions with different retention times in a segmented manner, performing high performance liquid analysis by rotary drying inspection, retaining fractions with the peak appearance time consistent with that of a target component in a liquid chromatography analysis positioning chart of the bulk drugs, and concentrating and drying under reduced pressure;
3) second preparation
Dissolving the enriched sample powder obtained in the step 2) with 65% methanol, filtering the solution through a 0.45-micron microporous filter membrane, and performing secondary preparation to prepare a liquid chromatographic column filler with the particle size of 5 microns and the pore diameter of 100A, wherein the mobile phase is a mixture of a liquid chromatographic column filler and a liquid chromatographic column filler in a volume ratio of 70: 30 methanol/1 thousandth formic acid, the detection wavelength is 210nm, the elution time is 85min, the flow rate is 10mL/min, the sample injection amount is 0.5mL, the sample is continuously injected through a trocar,
receiving component peak fractions with different retention times in segments, performing high performance liquid analysis by rotary drying inspection, retaining fractions with peak emergence time consistent with that of target components in a liquid chromatography analysis positioning chart of the bulk drugs, and concentrating and drying under reduced pressure.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101945884A (en) * | 2008-02-27 | 2011-01-12 | 花王株式会社 | Agent for preventing or ameliorating skin aging |
| CN102532241A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Method for purifying sodium aescinate |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101945884A (en) * | 2008-02-27 | 2011-01-12 | 花王株式会社 | Agent for preventing or ameliorating skin aging |
| CN102532241A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Method for purifying sodium aescinate |
Non-Patent Citations (2)
| Title |
|---|
| New saponins from the seeds of Aesculus chinensis;Zhang zhizhen;《Chemical and Pharmaceutical Bulletin》;19991130;第47卷(第11期);第1515-1520页 * |
| 娑罗子中七叶皂苷类成分的最新研究进展;熊艳等;《中药材》;20160531;第39卷(第5期);第1195-1199页 * |
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