CN1813893B - Method for extracting effective portion of polyphenol acid - Google Patents
Method for extracting effective portion of polyphenol acid Download PDFInfo
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- CN1813893B CN1813893B CN 200510129368 CN200510129368A CN1813893B CN 1813893 B CN1813893 B CN 1813893B CN 200510129368 CN200510129368 CN 200510129368 CN 200510129368 A CN200510129368 A CN 200510129368A CN 1813893 B CN1813893 B CN 1813893B
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Abstract
The present invention discloses a method for extracting polyphenolic acid active constituent, said method includes two steps: firstly, adopting absolute methanol or methanol aqueous solution whose concentration is greater than 95% to make extraction, then utilizing macroporous resin to make separation. The main components of obtained polyphenolic acid active constituent are salviolic acid B and its magnesium salt and ammonium potassium salt, their content is 73-75% of total weight, and the secondary components are caffeic acid trimer and dimmer, their content is 12-14% of total weight.
Description
Technical field
The present invention relates to from Chinese medicine, extract the method for effective site.Specifically, relate to the method for from salvia, extracting effective portion of polyphenol acid, relate more specifically to from Radix Salviae Miltiorrhizae, extract the method for effective portion of polyphenol acid.
Technical background
Traditionally, Radix Salviae Miltiorrhizae is a kind of commonly used effective Chinese medicine that is used for the treatment of as cardiovascular diseasies such as angina pectoris, hypertension, coronary heart disease and apoplexy.But general usefulness all is the product of slightly carrying that extract from Radix Salviae Miltiorrhizae, or directly cuts into slices with Radix Salviae Miltiorrhizae.Nearly two during the last ten years, and experiment in vitro is found the effective ingredient in the Radix Salviae Miltiorrhizae, mainly is polyphenol acid compounds, particularly salvianolic acid B and its esters.Results of in vitro studies shows, salvianolic acid B and its esters, except being used for the treatment of cardiovascular disease (Chinese patent CN02155004,01110378 and 98111076) outside, the potentiality that also have other multiple disease of treatment, as chronic hepatopathy (Chinese patent CN 99113644), tumor (Chinese patent CN 02136970 and 94103933), peptic ulcer (Chinese patent CN 98114172), (U.S. Pat 6043276 such as hepatitis B (Chinese patent CN 95105902) and acquired immune deficiency syndrome (AIDS), Chinese patent CN 95105902 and 99809677, list of references Antiviral Research 55:91-96,2002).Plant from other Salvia, in Coleus parvifolius and Cordia spinescens, isolated salvianolic acid B and its esters, experiment in vitro proves that equally they have the effect of anti AIDS virus (list of references Phytotherapy Research 17:232-239,2003 and 11:490-495,1997).
Along with the continuous discovery of the new purposes of polyphenol acid compounds in the Radix Salviae Miltiorrhizae, the patent of separation and purification polyphenol acid compounds preparation method is also in continuous increase.These methods are broadly divided into two types: (one) earlier with hot water lixiviate Danshen Root, leach water miscible polyphenol acid after, again through the distinct methods purification.Acid is carried and is separated (Chinese patent CN02160771) with macroporous resin as reuse; With macroporous resin chromatography and ethanol refining (Chinese patent CN 98111076); Acid is carried, Sephadex LH20 and high pressure liquid chromatography (HPLC) (Chinese patent CN 99809677); Or again through silica gel resin and high pressure liquid chromatography (HPLC) (Chinese patent CN98114172).(2) replace hot water lixiviate Danshen Root with the aqueous solutions of organic solvent of low concentration, leach water miscible polyphenol acid after, again through the distinct methods purification.As with 50-80% acetone water or 60-95% ethanol water to the Danshen Root lixiviate, the resin that reuse is different or certain resin liquid chromatography (LC) (Chinese patent CN 02160771,02137375 and 95113971) that adds high pressure is carried out purification.
Purpose of the present invention is to set up that a kind of technology is simple, cost is low and product quality is high and the method for stable and controllable, from salvia particularly the Radix Salviae Miltiorrhizae separation and purification go out a kind of effective portion of polyphenol acid, satisfy the demand in market.
Summary of the invention
The present invention is with a kind of simple two step method, from salvia particularly the Radix Salviae Miltiorrhizae separation and purification go out a kind of polyphenol acid position.Because preparation method is very simple, so good reproducibility as a result, the quality complete stability of compositions is controlled.Method of the present invention comprises following two steps:
(1) plant powder extracts:
Plant powder extracts greater than 95% methanol aqueous solution with absolute methanol or concentration, removes methanol after extracting fully, and precipitation is removed in the water-soluble back centrifugal filtration of residue;
(2) column chromatography:
Filtrate behind an amount of washing post, is used the methanol aqueous solution eluting through macroporous resin column chromatography, and pressure reducing and steaming first alcohol and water promptly gets effective portion of polyphenol acid after the residue dried.
Describedly extract greater than 95% methanol aqueous solution, be preferably with absolute methanol and extract with absolute methanol or concentration.
Described extraction comprises infusion process or percolation.
The gained effective portion of polyphenol acid, through high pressure liquid chromatography (HPLC), high-efficient silica gel book plate layer chromatography, analysis such as colorimetric and mass spectrum, the main component of finding it is salvianolic acid B and magnesium salt and ammonium potassium salt, and their structural formula accounts for the 73-75% (as shown in Figures 2 and 3) of gross weight as shown in Figure 1, their molecular weight is respectively 718,740 and 773 (as Fig. 4 and shown in Figure 6).Submember is caffeic acid trimer and dimer, and both account for 12-14% (as shown in Figures 2 and 3) altogether.According to mass spectral analysis, the caffeic acid trimer is based on the polyphenol acid (as shown in Figure 7) of molecular weight 520 and 516; Dimer is 296 polyphenol acid (as shown in Figure 8) based on molecular weight.
Under same experimental conditions, the effective portion of polyphenol acid to from four different time preparations has compared their high pressure liquid chromatography (HPLC) and high-efficient silica gel book plate layer chromatography.The result shows no matter four batches of effective portion of polyphenol acid are their high pressure liquid chromatography (HPLC)s, or high-efficient silica gel book plate layer chromatography collection of illustrative plates, all quite similar each other (as Fig. 9 and shown in Figure 10).Explanation is with the effective portion of polyphenol acid of the present invention preparation, good reproducibility as a result, and the quality complete stability of compositions is controlled.
The preparation method difference of the present invention and prior art is, breaks the normal procedure, and the first step replaces hot water or the lower aqueous solutions of organic solvent of concentration that prior art adopted, lixiviate plant powder with absolute methanol or concentration greater than 95% methanol aqueous solution.Experiment shows, with hot water or the lower aqueous solutions of organic solvent lixiviate 100 gram Danshen Roots of concentration that prior art adopted, extractum can reach 50-60 gram, and with absolute methanol or concentration greater than 95% methanol aqueous solution lixiviate, extractum has only about the 20-30 gram.Because the hydrophobicity of methanol is low, this change both can have been leached water miscible polyphenol acid, can reduce in a large number again that some other material soluble in water enters lixiviating solution and disturbs the purification of back in the Radix Salviae Miltiorrhizae.Therefore, carry out a step macroporous resin chromatography again, just can obtain the effective portion of polyphenol acid of more enrichment, its main component is salvianolic acid B and magnesium salt and ammonium potassium salt, accounts for the 73-75% (salvianolic acid B is the caffeinic tetramer) of gross weight; Submember is caffeinic trimer and dimer, and both account for 12-14% altogether.
Description of drawings
Fig. 1 is the molecular weight and the structural formula of salvianolic acid B and magnesium salt and ammonium potassium salt, wherein,
Salvianolic acid B 718 R=HH
Magnesium salt 740 R=Mg
Ammonium potassium salt 773 R=NH
4K
Fig. 2 is the high pressure liquid chromatography figure of the effective portion of polyphenol acid of embodiment 1.
Fig. 3 is the product of embodiment 1 and the high-efficient silica gel book plate layer chromatography spectrum of the fraction in the test example 17,6 and 5.
Fig. 4 is mass spectrum (ES-MS) figure of the effective portion of polyphenol acid of embodiment 1.
Fig. 5 is the pure salvianolic acid B of contrast usefulness and the mass spectrum of its esters.
Fig. 6 is the mass spectrum of the separating obtained fraction 7 of effective portion of polyphenol acid high pressure liquid chromatography of embodiment 1.
Fig. 7 is the mass spectrum of the separating obtained fraction 6 of effective portion of polyphenol acid high pressure liquid chromatography of embodiment 1.
Fig. 8 is the mass spectrum of the separating obtained fraction 5 of effective portion of polyphenol acid high pressure liquid chromatography of embodiment 1.
Fig. 9 is the high pressure liquid chromatography figure of four batches of extracts.
Figure 10 is the high-efficient silica gel book plate layer chromatography figure of four batches of extracts.
The specific embodiment
The embodiment that provides below is only for further specifying the present invention, and they are to the present invention and do not constitute any limitation.
Embodiment 1
The first step, absolute methanol extracts:
In 10 kilograms of Danshen Roots, add absolute methanol and soak three times, add 60,50 and 40 liters successively, soaked 8 hours at every turn, merge three times leachate.Methanol in the pressure reducing and steaming leachate adds 30 premium on currency, make residue dissolving after, filtrate is collected in centrifugal filtration (4000 rev/mins).
Second step, column chromatography:
Filtrate is through the AmberliteXAD-4 column chromatography.The chromatographic column diameter is 30 centimetres, and is high 80 centimetres, in adorn 20 kilograms of resins.Nearly 30 liters of filtrates slowly add in the post, and water, 20% and 80% methanol aqueous solution are washed post, each 150 liters successively.Desired effective portion of polyphenol acid is eluted in 80% methanol aqueous solution.Pressure reducing and steaming first alcohol and water further promptly obtains 0.22 kilogram of light brown effective portion of polyphenol acid after the drying, is 2.2% by the Radix Salviae Miltiorrhizae yield.More than two the step, except that the distillation, all at room temperature the operation.
The high pressure liquid chromatographic analysis of test example 1 effective portion of polyphenol acid
The product of embodiment 1 is carried out high pressure liquid chromatographic analysis, and employing reversed-phase column C18 (10 * 250mm), sample size 125 micrograms, with methanol-water-trifluoracetic acid eluting, volume ratio is 4: 6: 0.05%, flow velocity is 1.5ml/min.The results are shown in Figure 2, wherein, fraction 5 and 6 is based on caffeic acid dimer and trimer; Fraction 7 is salvianolic acid B and magnesium salt and ammonium potassium salt.And through the weighing method evaluation, fraction 5 and 6 accounts for 13.1% of total sample size altogether, and fraction 7 accounts for 75.1%.
The high-efficient silica gel book plate layer chromatography of test example 2 effective portion of polyphenol acid is analyzed
Product and the fraction in the test example 17,6 and 5 of embodiment 1 are carried out high-efficient silica gel book plate layer chromatography.Point sample is in proper order: 1. the product of embodiment 1; 2. fraction 5; 3. fraction 6; 4. fraction 7; 5. pure salvianolic acid B of reference substance and magnesium salt thereof.Launch to adopt chloroform-acetone-formic acid (volume ratio=8: 8: 1).Chromatoplate is sprayed with the phosphoric acid that contains 8%, 3% Schweinfurt green aqueous solution, 200 ℃ of heating carbonizations colour developings.Fig. 3 is high-efficient silica gel book plate layer chromatography result.Among the figure, a and b represent the shift position based on the mixture of caffeic acid trimer and dimer respectively.C represents the position that salvianolic acid B and magnesium salt and ammonium potassium salt thereof move.
The mass spectrum (ES-MS) of test example 3 effective portion of polyphenol acid is analyzed
Adopt anion reflection pattern.Sample is an aqueous solution, about 5 micrograms of sample feeding amount.Fig. 4 is the mass spectrum of effective portion of polyphenol acid.Among the figure, the structural formula of salvianolic acid B such as top right plot, the tetramer of genus coffea acid, molecular weight are 718, so m/z717 is from salvianolic acid B; M/z739 and 771 are respectively from the magnesium salt and the ammonium potassium salt of salvianolic acid B.Ion m/z519 and near less m/z559 thereof, 515 and 493, be caffeinic trimer all from belonging to, what they had may just be present in the extract originally.When carrying out mass spectrum, degraded and polymerization can take place in molecule, and what therefore have may might remove a hydration caffeic acid from salvianolic acid B as m/z519 from salvianolic acid B and its esters degradation product.Ion m/z321,339 and 295, be caffeinic dimer all from belonging to, as trimer, what they had may just be present in the extract originally, and what have may be from three and the fragment that generates when measuring of tetramer molecule.Near m/z1457 ion is Salvianolic acid B's a polymer, is exactly polymer from salvianolic acid B and magnesium salt thereof as m/z1457.44 is CO among the figure
2Molecular weight, the 16th, the atomic weight of O.
The mass spectrum that test example 4. contrasts with pure salvianolic acid B and its esters
Experiment condition the results are shown in Figure 5 with test example 3.Visible salvianolic acid B and magnesium salt and ammonium potassium salt quasi-molecular ions among the figure, the also visible simultaneously molecular fragment caffeic acid trimer that their produce when measuring, the quasi-molecular ions of dimer and their polymer.These quasi-molecular ions are consistent basically with the corresponding quasi-molecular ions of above-mentioned composition.Quantitative analysis results shows that salvianolic acid B and magnesium salt thereof and ammonium potassium salt account for 75.1% of gross weight in the extract, and caffeic acid trimer and dimer account for 13.1% altogether, and mass spectrometry results and its coincide herein.Other explanation is with test example 3.
The mass spectral analysis of test example 5 fractions 7
Experiment condition the results are shown in Figure 6 with test example 3.Visible salvianolic acid B and magnesium salt and ammonium potassium salt quasi-molecular ions among the figure, their molecular fragment caffeic acid trimer, the quasi-molecular ions of dimer and their polymer, all with the mass spectrum of pure salvianolic acid B of Fig. 5 and its esters on corresponding quasi-molecular ions almost consistent.Show and slip to divide 7 to form by salvianolic acid B and magnesium salt thereof and ammonium potassium salt.Other explanation is with test example 3.
The mass spectral analysis of test example 6 fractions 6
Experiment condition the results are shown in Figure 7 with test example 3.Wherein, ion m/z519,521 and 515 all belong to the caffeic acid trimer.Ion m/z519 removes a hydration caffeic acid molecule from salvianolic acid B; M/z521 may be from the saturate of ion m/z519; M/z515 may be from the sodium salt (494+22-1) of salvianolic acid A.During high pressure liquid chromatography (HPLC), slip and divide 6 and 7 to have a bit overlapping, so the quasi-molecular ions of the visible salvianolic acid B magnesium of Fig. 7.Ion m/z359 and 321 belongs to the caffeic acid dimer, the molecular fragment that the caffeic acid three and the tetramer produced when they may come comfortable mensuration, and they can not separate fully with trimer during high pressure liquid chromatography (HPLC) under this experiment condition.
The mass spectral analysis of test example 7 fractions 5
Experiment condition the results are shown in Figure 8 with test example 3.Wherein, ion m/z379,335 and 295 all belong to the caffeic acid dimer, and ion m/z379 may be from the sodium salt of preceding alkannic acid, and m/z335 is the methyl ester of m/z321, and m/z295 removes a CO from salvianolic acid G
2As aforementioned, ion m/z559,521,515 and 493 all belong to the caffeic acid trimer, may be under this experiment condition during high pressure liquid chromatography (HPLC), they and can not separate fully with dimer.
The high pressure liquid chromatographic analysis of 8 four batches of extracts of test example
Fig. 9 is the high pressure liquid chromatography figure of four batches of extracts of different time, wherein, A, B, C represents respectively that with D sample is from four different lot numbers.Here, when carrying out chromatography, 0 to 30 minute, after instrumental sensitivity is 0.02,30 minute, change 0.1 into, the same peak height of this expression, with 30 minutes after compare, be exaggerated 5 times before 30 minutes.Other explanation is as Fig. 2.
The high-efficient silica gel book plate layer chromatography of 9 four batches of extracts of test example
Figure 10 is the high-efficient silica gel book plate layer chromatography of above-mentioned four batches of extracts, and wherein, 1 to 4 sample is from four the different lot numbers identical with Fig. 8; The 5th, pure salvianolic acid B of reference substance and magnesium salt thereof.The point sample amount all is about 30 micrograms.Other explanation is as Fig. 3.
Heavy metal, methanol and chloroform residuals content are measured in test example 10 effective portion of polyphenol acid
According to the requirement of the Chinese Pharmacopoeia of version in 2000, measured the content of heavy metal, methanol and chloroform residue in the effective portion of polyphenol acid that obtains among the embodiment 1, find that they all are lower than the content limit value of state-set standard.
Assay is as follows:
| Project | Pharmacopeia requires (ppm) | Check result (ppm) |
| Heavy metal | ≤10 | ≤10 |
| Methanol | ≤3000 | ≤140 |
| Chloroform | ≤60 | ≤50 |
Concrete condition determination and algoscopy are as follows:
The condition determination of solvent residual amount:
Instrument is Agilent 6890 gas chromatograpies, fid detector; Chromatographic column is DB624 (6% cyanogen propylbenzene-94% methylsiloxane base co-polymer, 30m * 0.53mm * 3 a μ m) chromatographic column; Carrier gas is a nitrogen; Column flow rate is nitrogen 2.0ml/min (constant current); Split sampling (split ratio 1: 1); Column temperature: service routine heats up, and initial temperature is 50 ℃, keeps 5 minutes, and the heating rate with 50 ℃ of per minutes rises to 80 ℃ then, keeps 6 minutes, and the heating rate with 50 ℃ of per minutes rises to 200 ℃ then, keeps 4 minutes; Injector temperature is 180 ℃; Detector temperature is 260 ℃; Sample size is 1.0 μ l.
The algoscopy of solvent residual amount is:
Precision takes by weighing methanol, each is an amount of for chloroform, add dimethyl sulfoxine quantitatively dilution make the solution product in contrast that contain methanol 150 μ g, chloroform 3 μ g among every 1ml respectively approximately; Other gets the about 0.5g of this product, and accurate the title decides, and the accurate dimethyl sulfoxine 10ml that adds makes dissolving, shakes up, as need testing solution.Precision is measured each 1 μ l of above-mentioned two kinds of solution, with DB624 (6% cyanogen propylbenzene-94% methylsiloxane base co-polymer, 30m * 0.53mm * 3 μ m) chromatographic column, measure in accordance with the law, the record chromatogram, each is organized peak-to-peak separating degree and should meet the requirements, and presses external standard method with calculated by peak area, methanol≤3000ppm, chloroform≤60ppm.
The measuring method of heavy metal is: get the residue of leaving under the residue on ignition item, according to Chinese Pharmacopoeia two appendix VIIIH of version in 2000 (second method) method inspections down.The contrast solution made from standard lead solution (10mg/ml) 1ml compares.The color of need testing solution all be shallower than the contrast liquid (≤10ppm).
Methanol aqueous solution with 98% extracts:
In 10 kilograms of Danshen Roots, slowly add 150 liter 98% methanol aqueous solution, extract through percolation.Obtain after the extracting solution, comprise the operation in second step, all with embodiment 1.After testing, the main component of gained effective portion of polyphenol acid is salvianolic acid B and magnesium salt and ammonium potassium salt, accounts for 74.0% of gross weight, and submember caffeic acid trimer and caffeic acid dimer account for 13.2% of total amount altogether.By this routine yield of Radix Salviae Miltiorrhizae is 2.3%.
Methanol aqueous solution with 95% extracts:
This example replaces the absolute methanol except that the methanol aqueous solution with 95%, and other is operated all with embodiment 1.After testing, the main component of gained effective portion of polyphenol acid is salvianolic acid B and magnesium salt and ammonium potassium salt, accounts for 73.7% of gross weight, and submember caffeic acid trimer and caffeic acid dimer account for 12.5% of total amount altogether.By this routine yield of Radix Salviae Miltiorrhizae is 2.1%.
Claims (5)
1. from the plant Radix Salviae Miltiorrhizae, extract the method for effective portion of polyphenol acid, it is characterized in that: comprise following two steps:
(1) Danshen Root extracts:
Danshen Root extracts greater than 95% methanol aqueous solution with absolute methanol or concentration, removes methanol after extracting fully, and precipitation is removed in the water-soluble back centrifugal filtration of residue;
(2) column chromatography:
Filtrate behind an amount of washing post, is used the methanol aqueous solution eluting through macroporous resin column chromatography, and pressure reducing and steaming first alcohol and water promptly gets effective portion of polyphenol acid after the residue dried.
2. the method for extraction effective portion of polyphenol acid as claimed in claim 1, wherein said extraction comprises infusion process or percolation.
3. the method for extraction effective portion of polyphenol acid as claimed in claim 1 or 2 is characterized in that: the main component of extracting the gained effective portion of polyphenol acid is salvianolic acid B and magnesium salt and ammonium potassium salt, accounts for the 73-75% of gross weight; Submember is caffeinic trimer and dimer, and both account for 12-14% altogether.
4. the method for extraction effective portion of polyphenol acid as claimed in claim 1 or 2, wherein said macroporous resin comprise Amberlite XAD series, Diaion HP series and homemade non-polar macroporous resin.
5. the method for extraction effective portion of polyphenol acid as claimed in claim 4, wherein said homemade non-polar macroporous resin is GDX-104.
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| CN 200510129368 CN1813893B (en) | 2004-12-06 | 2005-12-06 | Method for extracting effective portion of polyphenol acid |
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|---|---|---|---|
| CN 200410101047 CN1654067A (en) | 2004-12-06 | 2004-12-06 | Method for extracting effective site of phenolic acid |
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| CN 200510129368 CN1813893B (en) | 2004-12-06 | 2005-12-06 | Method for extracting effective portion of polyphenol acid |
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| CN1436553A (en) * | 2002-02-08 | 2003-08-20 | 余琛 | Method of extracting effective component in red sage |
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